bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative analysis of the conservation of nucleoprotein immunogenic T-cell epitopes of master donor viruses for live and inactivated influenza vaccines
Authors: Rak Alexandra Ya. , Rudenkо L. G. , Isakova-Sivak I. N.
Published: July 2024

Antigen-specific T cells are an important part of antiviral responses, and modern influenza vaccines are designed to induce this mode of immunity. Live attenuated influenza vaccine (LAIV) is a potent inducer of T-cell immunity because of its ability to cause productive infection in the upper respiratory tract. Inactivated influenza vaccines (IIV) and novel vaccine candidates can also induce virus-specific T-cells when appropriate adjuvants are used. In this case, non-structural and intrinsic antigens of the master donor viruses, particularly nucleoprotein (NP), are the main targets for the development of T-cell immunity. The most commonly used donor strains for LAIVs and IIVs worldwide were derived from viruses isolated between 1933 and 1960. In this regard, the question of conservation of epitopes immunogenic for CD8⁺ T-lymphocytes (CTL-epitopes) in donor-derived NPs, i.e., the ability of cytotoxic T cells specific to the donor’s NP to recognize modern influenza A virus nucleoproteins, is relevant. The aim of the study was to evaluate the conservation of CTL-immunogenic NP epitopes of donors traditionally used to create LAIVs and IIVs. Materials and methods. Epitope NP analysis was performed for 1614 and 1767 strains of influenza A virus subtypes H1N1 and H3N2, respectively, which circulated in 2009–2023 (data from the NCBI Influenza Virus Database). Immune Epitope Database (IEDB, www.iedb.org), NetCTL’s built-in CTL-epitope prediction algorithm and NetChop proteolysis site predictor were used. CTL-epitopes were mapped to NPs of master donor viruses A/Leningrad/134/17/57 (H2N2), A/Ann Arbor/6/60 (H2N2), A/PR/8/34 (H1N1), and A/WSN/1933 (H1N1) using the CrustalO alignment algorithm in JalView 2.8.1 Software. The immunogenicity and conservation of selected epitopes were further evaluated using IEDB T-cell Immunogenicity Predictor and Epitope Conservancy Assay, respectively. Results. The majority of immunogenic CTL-epitopes of donor viruses proved to be non-conserved, i.e., not found in NPs of circulating influenza strains. Conversely, most CTL-immunogenic NP epitopes of modern viruses are absent in donor viruses and cannot be induced by vaccination with conventional vaccines. The data obtained indicate the need to actualize NP in vaccine composition by directed mutagenesis of the donor-derived NP gene or by introduction of the gene encoding NP of circulating influenza viruses into vaccine strains.
Comparative assessment of interferon activity in influenza and COVID-19
Authors: Ospelnikova Tatiana P. , Svitich O. A. , Ershov F. I.
Published: July 2024

Among respiratory viruses, the most serious complications are caused by influenza A and B viruses, as well as coronaviruses. Most studies determined the absolute content of interferons (IFNs) of different types in blood serum. However, serum IFN protein concentrations do not always reflect the level of antiviral protection. The purpose of this study was a comparative assessment of interferon status in patients with ARVI: influenza and the acute stage of COVID-19. Materials and methods. We used biomaterial in the form of whole blood samples from 113 patients with influenza and 110 patients in the acute phase of moderate COVID-19. The body’s antiviral defense during ARVI was assessed by determining the activity of type I and II interferons produced by blood leukocytes using the “Interferon status” method in a cell-virus system simulated in vitro. Results. This work reveals a statistically significant decrease in the biological activity of interferons produced by blood leukocytes in influenza and a deficiency of IFN activity in COVID-19, compared with reference values, and also shows possible prospects for the treatment of these nosologies with such immunoactive drugs as IFN inducers (cycloferon, Kagocel) and immunomodulators (ingavirin, multicomponent vaccine Immunovac-VP-4). Conclusion. The results of IFN activity are necessary to assess the antiviral potential of the body, especially with COVID-19, given the “novelty” of the infection, the severity and variety of its clinical manifestations. Today it is known that the SARS-CoV-2 virus is capable of penetrating not only into the epithelial cells of the upper respiratory tract, epithelial cells of the stomach and intestines, but also into the cells of the esophagus, heart, adrenal glands, bladder, brain, as well as into the vascular endothelium and macrophages. Coronavirus SARS-CoV-2 inhibits the expression of cellular genes, including innate immune genes, and has a negative effect on the IFN system. The use of IFN inducers and immunomodulators for influenza and COVID-19 has shown immunological feasibility and clinical promise.
The role of сhemokine CX3CL1/Fractalkine in chronic viral hepatitis B
Authors: Arsentieva Natalia A. , Batsunov O. K. , Liubimova N. E. , Basina V. V. , Esaulenko E. V. , Totolian A. A.
Published: July 2024

Hepatitis B is an infectious disease resulting from infection with the hepatitis B virus. Chronic hepatitis B (CHB) is characterized by prolonged inflammation in the liver, the development of fibrosis, liver cirrhosis and hepatocellular carcinoma. Factors of the immune system play a critical role in the pathogenesis of CHB. Thanks to chemokines, immune cells migrate to the site of inflammation to implement their effector functions. The CX3CL1/Fractalkine chemokine is the only member of the CX3C family of chemokines with unique structural and functional properties. Its receptor CX3CR1 is expressed mainly on the surface of cytotoxic effector lymphocytes such as NK cells, TNK and cytotoxic T lymphocytes. The purpose of our study was to analyze the content of CX3CL1/Fractalkine in the blood plasma of patients with CHB and the analysis of this chemokine with liver fibrosis. The concentration of CX3CL1/Fractalkine was determined in the blood plasma of patients with CHB using a multiplex assay based on xMAP technology. Blood plasma from patients with chronic viral hepatitis C (CHC) and autoimmune liver diseases (AILD) was used as a comparison group. The control group consisted of healthy individuals. For statistical analysis of data, nonparametric statistics methods were used: Kruskal–Wallis test, Spearman correlation coefficient ROC-analysis. It was shown a reduced level of CX3CL1/Fractalkine in patients with CHB compared with the control group (p = 0.0003) and with the comparison groups of CHC (p 0.0001) and AILD (p = 0.0005). A reduced concentration of CX3CL1/Fractalkine was shown in the blood plasma of CHB patients with initial fibrosis (p = 0.0092) and severe fibrosis/cirrhosis (p = 0.0009), while in patients with severe fibrosis/cirrhosis, a significantly reduced level of this chemokine was established compared with the initial degree of liver fibrosis (p = 0.0081). Correlation analysis revealed a highly significant inverse relationship between the severity of liver fibrosis and the content of CX3CL1/Fractalkine in the blood plasma of patients with CHB (Spearman r = –0.33; p = 0.02). Thus, the chemokine CX3CL1/Fractalkine is included in the immunopathogenesis of CHB; its reduced content is characteristic only of CHB and does not change in other chronic liver diseases. It is involved in the processes of liver fibrosis during infection with the hepatitis B virus. The concentration of the chemokine CX3CL1/Fractalkine depends on the stage of liver fibrosis in CHB, and a decrease in the level of CX3CL1/Fractalkine in the blood plasma can serve as a negative factor in the development of CHB.
Conserved linear B-cell peptides among the influenza A viral neuraminidases enhance the cross-protective potential of inactivated whole-virion influenza vaccine
Authors: Kotomina Tatiana S. , Sychev I. A. , Rak A. Ya. , Wong P.-F. , Bazhina A. V. , Isakova-Sivak I. N. , Rudenko L. G.
Published: July 2024

Introduction. Influenza is a disease caused by a widespread virus with pandemic potential. Frequently, individuals vaccinated against seasonal influenza virus are still susceptible to the disease, indicating the need to improve the immunogenic potential of existing vaccines. To assess the efficacy of influenza virus vaccines, immune response only to a single viral antigen — hemagglutinin molecule, is taken into consideration. However, according to preclinical and clinical studies, neuraminidase (NA) stimulates cross-protective immunity, which is effective against not only homologous but also drifted variants of influenza A virus. Materials and methods. In the present study, we investigated the ability of previously selected conserved linear B-cell NA epitopes (SGYSGK, SWPDGK, EECSCYPK, VELIRGRK) to enhance the immunogenicity of an inactivated whole-virion influenza vaccine based on the model strain PR8 (iPR8). BALB/c mice were injected with iPR8 in combination with one of the peptides intramuscularly three times at two-week intervals. Blood samples were collected 14 days after the last immunization, after which the mice were challenged with heterosubtypic influenza viruses H1N1pdm09 and H3N2. Results. All immunized mice showed induction of H1N1 (PR8)-specific IgG antibodies two weeks after the third immunization. The group of mice immunized with the iPR8 vaccine preparation in combination with VELIRGRK peptide showed the most pronounced induction of IgG antibodies to the H6N1 reassortant strain, the NA of which corresponds to the iPR8 virus, indicating the ability of the NA peptide to stimulate the production of NA-specific antibodies. However, the antibodies produced after immunization were not capable to inhibit the NA enzymatic activity. Despite this, mice immunized with iPR8 in combination with anti-NA peptides showed a higher survival rate after infection with heterologous virulent influenza viruses: A/California/07/09 (H1N1pdm09) and A/Philippines/2/82 (H3N2) compared to the PBS and iPR8 groups. Conclusion. Thus, the study demonstrated the immune-potentiating effect of individual peptides corresponding to conservative linear epitopes of the NA molecule in combination with a standard inactivated influenza vaccine, which made it possible to improve the protective effect of the vaccine against heterosubtypic influenza viruses.
Characteristics of virus-specific immunological reactions following COVID-19 vaccination in heart transplant recipients
Authors: Zafranskaya Marina M. , Nizheharodava D. B. , Shatova O. G. , Russkih I. I. , Velichko A. V. , Vanslau M. I. , Novitskaya S. F. , Denisevich T. L. , Kolyadko M. G. , Kurlyanskaya E. K.
Published: July 2024

Heart transplant patients are at an increased risk of COVID-19 infection adverse outcomes because of underlying immunosuppression and concomitant comorbidities. To date, all large-scale randomized controlled trials for various COVID-19 vaccines have excluded solid organ transplant recipients. Therefore, the efficacy and safety of coronavirus infection prevention using COVID-19 vaccines in transplant heart patients has not been sufficiently studied. In this research, the evaluation of virus-specific immunological reactions after vaccination (double Vero Cell vaccination and Sputnik Light booster vaccination) in heart transplant patients has been carried out. In vaccinated heart transplant individuals who did not have a history of COVID-19, starting from 4–6 months after the 2nd dose of the vaccine, an increase in antibodies to S protein level of was observed, while maintaining statistically significant differences for 9–12 months after vaccination (regardless of whether with or without booster vaccination). However, the concentration of antibodies remained low, and 37% of patients detected no antibodies. In vaccinated heart transplant individuals following the previous COVID-19 infection, as compared to seronegative patients, post-vaccination immunity is accompanied by maintaining a high level of virus-specific IgG antibodies to the S protein of the SARS-CoV-2 virus in the dynamics of the post-vaccination period with a statistically significant increase of these antibodies by 9–12 months after booster vaccination. The specific cellular response (according to the assessment of CD3⁺154⁺ and CD3⁺IFNγ⁺ TNFα⁺ cells) to the S protein of the SARS-CoV-2 virus remained low throughout the entire follow-up period, was recorded in 5–40% of heart transplant patients and statistically significant changes in the number of spikereactive lymphocytes were observed in patients with a history of COVID-19 by 4–6 months after administration of the 2nd dose of the vaccine. This, together with the results of the assessment of the humoral response, indicates a more pronounced post-vaccination immunity in patients with a hybrid immunity. While developing a methodology for assessing the risk and benefit of a vaccination strategy for individual heart transplant patients, clinical efficacy, ongoing monitoring of rare serious adverse events, and data on vaccine immunogenicity should be taken into account.
Expression of pro-inflammatory cytokines (IL-18, IL-33) at the level of the mucous membrane, the entry point of infection, in persons who have experienced COVID-19 disease
Authors: Rasskazova Nadezhda D. , Abramova N. D. , Soshchenko T. D. , Kalyuzhnaya N. O. , Meremianina E. A. , Shatokhin M. N. , Zaitseva T. A.
Published: July 2024

Introduction. The mucous membrane of the upper respiratory tract is the entrance gate for a large number of infections, including the SARS-CoV-2 virus. That is why the main task of the immune system of the mucous membranes of the entrance gate of infection is to maintain respiratory function. High production of proinflammatory cytokines, which play a key role in the development of severe COVID-19 infection, leads to detrimental consequences for all body systems. Their long-term influence can not only aggravate chronic pathologies, but also significantly increase the recovery period, leading to a decrease in the quality of life of patients. Therefore, the purpose of this work is to study the expression of IL-18 and IL-33 genes at the level of the mucous membranes of the upper respiratory tract in patients who have had COVID-19 disease. Materials and methods. The present study involved patients who had moderate or severe COVID-19. The control group consisted of relatively healthy individuals. The expression levels of IL-18 and IL-33 were detected using RT-PCR. Results. During the entire period of rehabilitation after suffering from COVID-19, patients showed a tendency to increase the level of IL-18 expression at the level of the mucous membranes of the nasopharynx and oropharynx. The level of IL-33 production was also increased, but varied depending on the location and period of sample collection. Conclusion. This increase in the level of IL-18 during the rehabilitation period of patients after COVID-19 may be explained by the fact that the virus, by activating glia through olfactory receptor neurons, triggers a powerful immune response and promotes the production of a large number of pro-inflammatory cytokines. On the contrary, overexpression of IL-33 in the late stages of rehabilitation is most likely associated with its ability to restore barrier tissues of the mucous membranes of the upper respiratory tract. Thus, we can conclude that the virus promotes excessive production of pro-inflammatory cytokines, the amount of which increases maximally at the 6th month of rehabilitation after suffering from COVID-19.
Humoral immune response to shiga toxin 2 (Stx2) in children with escherichiosis with hemolytic-uremic syndrome
Authors: Shkuratova Maria A. , Khlyntseva A. E. , Kalmantaeva O. V. , Kartsev N. N. , Muzurov A. L. , Firstova V. V.
Published: July 2024

Shiga toxin-producing Escherichia coli (STEC) causes acute intestinal infections and also causes acute renal failure, especially in children. Shiga toxins (Stx) occupy a central place in the pathogenesis of hemolytic uremic syndrome (HUS) in Escherichiosis. The presented work analyzes the effectiveness of laboratory diagnostics of enterohemorrhagic escherichiosis in patients at the stage of manifestation of HUS and/or acute renal failure using microbiological, immunological research methods and PCR analysis. The study used clinical material from 30 patients in the pediatric intensive care unit of the St. Vladimir Children’s City Clinical Hospital in Moscow with symptoms of HUS aged from 8 months to 5 years. Blood sera from 20 healthy donors were used as control. As a result of PCR analysis, stx2 DNA was detected in 23.3% of cases. Bacteriological research made it possible to sow a pure culture of Escherichia coli O157:H7 in only 3.3% of cases. Since the development of HUS begins in patients with acute intestinal infection caused by Shiga toxin-producing microorganisms starting from the 5th day of the disease, when antibiotic therapy is already carried out, the bacteria can be completely destroyed, which makes it difficult to identify them by bacteriological methods, as well as to detect genes encoding Shiga toxin in PCR analysis. Typically, patients with HUS are admitted to the intensive care unit 5–7 days after the onset of the disease, when class G immunoglobulins specific to the pathogen are already beginning to circulate in the blood. In this regard, the use of immunological tests can be effective to confirm the diagnosis of STEC infection. In our studies, enzyme immunoassay allowed us to detect antibodies to Stx2A in 63.3% and to Stx2B in 43.3% of patients. Using immunoblotting, antibodies to Stx2A were detected in all sera obtained from patients and in 66.7% of cases to Stx2B. Immunoblot analysis was characterized by higher sensitivity for detecting antibodies to Stx2, however, due to the presence of an immunological layer among healthy people, it is preferable to use ELISA analysis. In healthy donors with antibodies to Stx2, the antibody titer was significantly lower than in patients. Laboratory confirmation of the diagnosis of STEC infection is difficult when conducting microbiological and molecular genetic studies, which is confirmed in this work. The effectiveness of laboratory diagnostics can be expanded by performing an ELISA aimed at detecting antibodies to Stx2A.
IL-6 and IL-18 cytokine traps in COVID-19
Authors: Korotaeva A. A. , Samoilova Elena V. , Chepurnova D. A. , Pogosova N. V. , Kuchiev D. T. , Paleev F. N.
Published: July 2024

Cytokines are mediators of immunity that regulate inflammation. Intensity of inflammatory process is strongly dependent on the cytokine type and duration of its effect. Interleukin 6 (IL-6) and interleukin 18 (IL-18) play an important role in the initiation and progression of inflammation. Cytokines regulate the inflammatory process in different ways by inducing or inhibiting inflammatory reactions. Functional activity of cytokines is limited by trap molecules whose levels determine initiation of protective or pathological effects of interleukins. Soluble glycoprotein sgp130 functions as a trap for IL-6, while IL-18 is controlled by IL-18 binding protein (IL-18BP). High IL-6 and IL-18 levels were recorded in COVID-19 patients, being associated with unfavorable outcome of the disease. Our objective was to compare sgp130 and IL-18BP levels in patients with different degrees of COVID-19. Retrospective study included 74 COVID-19 patients (40 men and 34 women) aged 63±14 years. The patients were assigned to groups according to severity of lung damage. Group 1 included patients without lung damage; group 2, patients with moderate pneumonia ( 50% lung damage); group 3, patients with severe pneumonia ( 50% lung damage). Plasma levels of cytokines and their trap molecules were determined by quantitative immunoenzyme assay. IL-6 and IL-18 plasma concentrations increased with COVID-19 severity. Ambiguous changes were recorded for their traps. Plasma levels of sgp130 were lower in patients with moderate pneumonia than in patients without lung damage. In patients with severe pneumonia sgp130 plasma concentrations were higher than those in patients with mild pneumonia, being similar to those in patients without lung damage. In contrast to sgp130, IL-18BP levels decreased with COVID-19 severity. Thus, an increase in IL-6 and IL-18 levels parallel to COVID-19 severity is accompanied by ambiguous changes in the levels of their trap molecules. The ratio between the levels of IL-6 and IL-18 and their traps reflects the degree of COVID-19 severity.
Impact of Tritrichomonas spp. on the immune system of Muc2–/– mice after antibiotic therapy
Authors: Goncharova E. P. , Bets Victoria D. , Makusheva Yu. S. , Litvinova E. A.
Published: July 2024

While pathogenic protists inhabiting the reproductive tract are well studied, the gastrointestinal (GI) tract contains a constitutive protist microbiota that is an integral part of the vertebrate microbiome. Currently, the effect of protozoan infections on the host immune system and their potential contribution to disruption of mucosal immune homeostasis are not well understood. Protists, along with bacteria and viruses, are permanent representatives of the human microbiota. The main attention of researchers is focused on studying their pathogenic role in gastrointestinal diseases. However, their role in symbiotic relationships with hosts is relatively little studied. It was previously shown that the closest human ortholog of mouse Trichomonas (Tritrichomonas spp.) is Trichomonas Dientamoeba fragilis, which can cause symptoms of inflammatory bowel disease. It is currently unclear whether Dientamoeba fragilis and other protist species such as Enteromonas spp., Entamoeba dispar are commensals, pathobionts, or pathogens of the human intestinal tract. Thus, information about the mutualistic relationships between protists, the gastrointestinal microbiota, and the immune system of mice can be used to understand host-protozoan relationships in humans. The data obtained allow us to evaluate the potential contribution of commensal protozoa in the formation of protective mechanisms of the mucous membrane of animals and humans. We have previously shown that antibiotic therapy leads to an increase in the number of Tritrichomonas spp. along with a reduction in bacteria in the gut of mice with a mutation in the Muc2 gene. A mutation in this gene leads to impaired formation of the intestinal mucosa in mice. Mice with a mutation in the Muc2 gene can be used as model to study human inflammatory bowel diseases (IBDs). In this work, we conducted a comparative study of the immunological status of Muc2–/– mice harboring Tritrichomonas spp. after antibiotic therapy for 2 weeks followed by gavage of Lactobacillus johnsonii into mice and mice without introduction of probiotic microorganisms (self-recovery). Analysis of the main populations of lymphocytes in the blood, spleen and lymph nodes showed that the introduction of Lactobacillus johnsonii after antibiotic therapy leads to a significant increase in the population of T-lymphocytes in the blood and spleen, and an increase in the number of helper T cells in the lymph nodes of Muc2–/– mice compared to mice without the addition of probiotic microorganisms.
Laboratory blood markers in COVID-19 and their connection to Viral variant
Authors: Korobova Zoya R. , Liubimova N. E. , Arsentieva N. A. , Zhebeleva M. S. , Chernykh E. I. , Davletshina V. V. , Kashchenko V. A. , Totolian A. A.
Published: July 2024

Morality rates in COVID-19 are dependent on timely diagnosis. Therefore, studying the relationship between laboratory markers and the severity of disease is important. The first wave of COVID-19 associated with the spread of the original strain of SARS-CoV-2, showed higher mortality rates caused by cytokine storm. As the viral variant changed, a change in the disease course towards a less pronounced inflammatory reaction was observed. These changes affected major players of inflammation, cytokines. However, cytokines are not the only markers in the inflammatory response. The purpose of this work was to determine the significance of laboratory markers in inflammation: WBC, C-reactive protein, ferritin, fibrinogen, and D-dimer. The study included 227 patients with acute COVID-19 in the first 5–7 days from the onset of the disease from January 2021 to March 2022. When compared with reference, all groups were characterized by reduced absolute values of lymphocytes. Correlation analysis between the absolute value of lymphocytes and plasma cytokine concentrations also revealed statistically significant strong relationships with the level of the chemokine CCL22/MDC. Given that CCL22/MDC is an important component of lymphopoiesis, its low concentrations may indicate dysregulation of this process in COVID-19. In addition, we noted a positive correlation between the level of C-reactive protein and IL-6 in peripheral blood. IL-6 is a proinflammatory cytokine, and its elevated levels have been associated with the development of severe COVID-19. One of its functions is the induction of C-reactive protein, and this trend persists regardless of which variant causes COVID-19. We also noted positive correlations between the concentrations of fibrinogen and IL-18, ferritin and IL-6, IL-18. Both of these proteins are involved in inflammation along with cytokines. The literature provides data on the significance of these markers for determining the severity of COVID-19. There is evidence of a synergistic effect of ferritin and IL-18 against viral pathogens. Of interest was the negative correlation between plasma D-dimer levels and IFNα. At the same time, data on the role of the latter in thrombus formation processes are increasingly appearing in the literature.
Long non-coding RNAs — regulators of rubella virus infection and antiviral response
Authors: Gulimov M. K. , Kalyuzhnaya N. O. , Ammour Yulia I. , Zverev V. V. , Svitich O. A.
Published: July 2024

Introduction. Rubella virus is an RNA-containing virus capable of infecting human cells and causing infectious disease. Infection of pregnant women with rubella virus can lead to abortion or congenital rubella syndrome (CRS), a set of long-term birth defects including incomplete fetal organ development and mental retardation. There is no specific treatment for rubella and CRS. The regulation of antiviral immune response and viral reproduction by long non-coding RNAs is currently under active investigation. In this study, we evaluated the changes in the expression profile of long non-coding RNAs in rubella virus-infected A549 epithelial by RNA sequencing. Materials and Methods. A549 cells were infected with a wild-type variant of laboratory strain C-77 of rubella virus with a multiplicity of infection of 1.0 infectious units per cell and incubated for 72 hours. Virus titres were determined by the CCID method in the sensitive RK-13 cell culture. 48 h after infection, the cell monolayer was lysed, RNA was isolated, and libraries were prepared for sequencing. Sequencing was performed on the NextSeq500 platform (Illumina, USA) in paired-end reading mode. Validation of the obtained RNA sequencing data was performed using quantitative real-time PCR. Results. Rubella virus replication affects the production of some long non-coding RNAs by altering their expression profile. Thus, upon infection of A549 epithelial cells with rubella virus, there was a significant increase in the expression of such long non-coding RNAs as GAS5, NEAT1, LUCAT1, MIR210HG, MEG3, EPB41L4A-AS1, ZFAS1, and SNHG 1, 7, 12, 29, 32. DANCR, IGFL2-AS1, IGFL2-AS1, MIR1915HG, and SNHG14 were most significantly decreased in expression. Gene ontology (GO)-analysis revealed that long non-coding RNAs are involved at different levels in the mechanisms of immune response, in particular, RNA processing and nucleic acid metabolism; therefore, up- and down-regulation of these molecules leads to modulation of human antiviral immune response in response to rubella virus infection. Conclusion. Thus, the regulation of long non-coding RNA production by rubella virus has been shown for the first time. Differentially expressed long non-coding RNAs can be used as prognostic and diagnostic biomarkers of viral diseases.
Longitudinal tracking of T-cell repertoire reveals long-lasting CD4⁺ yellow fever specific clone cluster
Authors: Salnikova Mariia A. , Lebedev Yu. B.
Published: July 2024

Infection is inconceivable without T cells. T cells not only eliminate virus-infected cells and participate in the formation of immunological memory, but also indirectly modulate the humoral response through the selection and maintenance of specific B cells. The T-cell receptor (TCR) recognizes processed antigen presented on the surface of cells in the MHC of one of two classes. Thus, the formed TCR repertoire reflects the history of encountered antigens through the prism of the specific organism with a particular set of MHC. To investigate changes in the TCR repertoire in response to acute viral infection, we utilized a yellow fever vaccination model. The yellow fever vaccine has been a benchmark for both safety and efficacy for over half a century. The vaccine is based on a live attenuated virus, allowing the study of the immune response under conditions closely to the viral infection. The yellow fever-specific T-cell response to immunodominant peptides presented on HLA-A02 is well studied, but experiments with HLA-A02-negative donors are still lacking. The aim of this study was to examine the dynamics of changes in the T-cell repertoire structure that occur in response to yellow fever vaccination in a donor without the HLA-A02 allele. We found that the overall T-cell response dynamics were similar to that in HLA-A02-positive donors: vaccination led to rapid expansion of yellow fever-reactive clones by day 14. Despite the absence of a known immunodominant epitope for HLA I alleles in this donor, the immune response also shifted towards CD8⁺ T cells, with increasing of the CD8⁺ clones fraction by day 53. The amino acid sequences of CDR3 TCRb yellow fever specific clones formed a stable cluster by CD4⁺ T cells, further confirming the presence of novel immunogenic epitopes.
Markers of CD4⁺ AND CD8⁺ T-cell exhaustion in hiv/hcv coinfected immunological non-responders to antiretroviral therapy
Authors: Saidakova Evgeniya V. , Korolevskaya L. B. , Vlasova V. V. , Shmagel N. G. , Shmagel K. V.
Published: July 2024

Coinfection with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) is a risk factor for immunological non-response to antiretroviral therapy. In cases of immunological non-response, HIV viral load suppression occurs without an increase in CD4⁺ T-cell counts, heightening the risk of morbidity and mortality in infected individuals. T-cell exhaustion may hinder their regeneration in immunological non-responders. This study aimed to identify markers of CD4⁺ and CD8⁺ T-cell exhaustion in HIV/HCV coinfected immunological non-responders. The study examined three clinical groups: 1) HIV/HCV coinfected immunological non-responders (CD4⁺ T-cells 350/µl blood; n = 9), 2) HIV/HCV coinfected individuals with a standard response to therapy (CD4⁺ T-cells 500/µl blood; n = 9), and 3) relatively healthy volunteers without HIV and HCV infections (n = 9). Ex vivo, the number of CD4⁺ and CD8⁺ T-cells expressing the inhibitory receptor PD-1 was determined using multi-color flow cytometry. In the 7-day in vitro experiment, cell cultures were stimulated with phytohemagglutinin. The number of dying proliferated CD4⁺ and CD8⁺ T-cells (CFSElowZombieUV+) was determined using multi-color flow cytometry. The amount of interleukin-2 in the culture supernatants was measured using an enzyme-linked immunosorbent assay. It was found that in HIV/HCV coinfected immunological non-responders, there was a higher number of CD4⁺ and CD8⁺ T-cells expressing PD-1, a phenotypic marker of exhaustion, compared to the other two groups. Furthermore, the frequency of dying dividing T-cells was higher in immunological non-responders, with an increase in CD4⁺ T-cells but not CD8⁺ T-lymphocytes. Similarly, a decrease in interleukin-2 production was found in stimulated T-cells of HIV/HCV coinfected immunological non-responders in the CD4⁺ T-cell pool, but not in CD8⁺ T-lymphocytes. Thus, in HIV/HCV coinfected immunological non-responders, CD4⁺ T-cells appear exhausted both phenotypically and functionally. While CD8⁺ T-cells express inhibitory receptors, they do not show functional impairments. It appears that the specialized therapy for HIV/HCV coinfected immunological non-responders should aim to improve CD4⁺ T-cell function.
Mice microglia cytokine profile changes under the influence of HSV-1
Authors: Bulgakova Irina D. , Zverev V. V. , Kravtsova E. O. , Usatova G. N. , Shoichet D. A. , Zadvornykh E. A. , Shumkina A. A.
Published: July 2024

Introduction. Today, prevalence of neurodegenerative diseases increases. In recent years, more studies have revealed a new knowledge about the role of microglia in the development of these diseases. Animal experiments showed that peripheral inflammation causes activation of microglia in brain. All this points to the essential role of the cells in the development of neurodegeneration. Under the influence of various factors, microglia can change the phenotype and participate in both repair and damage to brain cells. Chronic herpesvirus infection caused by HSV-1 is another known factor in the development of neurodegenerative pathology. However, the exact pathogenetic mechanisms are still unknown, nevertheless, studying the virus effect on microglia has great potential. The goal of our study in this connection was to assess the effect of HSV-1 on microglia polarization in mouse strain with normal susceptibility to this virus and in strain which is more resistant to the action of HSV-1. For this purpose, changes in the cytokine profile were detected. A comparison of interstrain differences in the expression of cytokine genes was also compared in control groups. Materials and methods. The study involved infecting C57BL/6 and BALB/c mice with herpes simplex virus type 1, an isolation of microglia was based on separation steps using a discontinuous gradient density, the cytokine profile was assessed by gene expression levels using a real-time reverse transcription PCR. To calculate the relative fold gene expression of samples the 2–ΔΔCt method was used. Statistical significance was determined using the Mann–Whitney U-test. Results. There were found no interstrain differences in cytokine gene expression in control groups of different mouse strains. At the same time, gene expression differed in the experimental groups: in BALB/c mice, the expression of genes for both pro-inflammatory and anti-inflammatory cytokines increased; in C57BL/6 mice, a slight increase in the expression of IL-1β genes was observed. Conclusion. The data indicate the formation of different microglial phenotypes after HSV-1 infection in different mouse strains. Apparently, in BALB/c mice there is a switch from the pro-inflammatory M1 phenotype of microglia to the anti-inflammatory M2 phenotype, while in C57BL/6 mice the attenuation of the infectious process occurs through a return to the original M0 phenotype.
Neutrophil extracellular traps as markers of infectious and aseptic inflammation
Authors: Kazimirskii Alexander N. , Salmasi J. M. , Poryadin G. V. , Panina M. I. , Kim A. E. , Rogozhina L. S.
Published: July 2024

One of the important problems of medicine is the search for new reliable markers of inflammation that would allow us to determine the type of inflammatory process. The aim of the study was to identify specific markers of infectious and aseptic inflammation based on the analysis of morphological variants of neutrophil extracellular traps (NETs). The study included 26 patients with various nosological forms of inflammation in the abdominal cavity (12 with acute appendicitis, 8 with acute cholecystitis, 6 with a diagnosis of pancreatitis/ pancreonecrosis) after surgery. The study also included 20 patients with post-COVID and a group of 10 volunteers no clinical manifestations of the disease, but at the same time had contacts with flu patients. Neutrophils were isolated using gradient centrifugation. Fluorescence microscopies with the dye SYBR Green (JSC “Evrogen”, Russia) were used for NET visualization and counting. NETs in the morphological form of neutrophils web structure were found in the blood of surgical patients with acute appendicitis who had a favorable course of the postoperative period. In a group of volunteers who were exposed to influenza but did not have symptoms of an infectious disease, we also found neutrophils web structure. In all patients with abdominal inflammation complicated by abdominal abscess, in addition to neutrophil web structure, abnormal forms of NETs were recorded, in particular, NETs in the form of single filaments. In the post-COVID patients an absolute absence of neutrophilis web structure was revealed. In all post-COVID patients, NETs are found only in the form of single filaments. The results of subsequent experiments showed that collagen peptides are inducers of the formation of NETs in the form of single filaments. The detection of neutrophils web structure is a sign of an infectious inflammatory process. NETs in the morphological form of single filaments are markers of aseptic inflammation, as well as a sign of endothelial damage.
Overexpressing miR-222-3p in cultured Mycobacterium Tuberculosis-infected macrophages does not affect their bacteriostatic activity
Authors: Shepelkova Galina S. , Evstifeev V. V. , Yeremeev V. V.
Published: July 2024

Tuberculosis, a disease caused by the bacterium Mycobacterium tuberculosis, is a major public health concern. Innate and adaptive immunity provide robust defense against pathogens. However, M. tuberculosis, which co-evolved with humans, has acquired many mechanisms to evade the immune response and ensure its intracellular existence and long-term survival within the host. Moreover, emerging evidence suggests that this secretive bacterium can alter expression of regulatory noncoding RNAs (including microRNAs), leading to dysregulation of biological processes underlying tuberculosis pathogenesis. For example, miR-222-3p has been shown to regulate the functional reprogramming of macrophages and is involved in the regulation of host innate immunity. Previously, we demonstrated the important role of miR-222-3p as a biological marker of tuberculosis activity. To confirm their biological targets and understand their role in the pathogenesis of tuberculosis, many research groups are working to establish functional relationships between miRNA expression under different conditions and their actual biological action using molecular biology and bioinformatics methods. In the present study, we demonstrated the effect of miR-222-3p overexpression on several functions of human macrophages of monocytic origin activated with M. tuberculosis antigens in in vitro culture. Specifically, we found that miR-222-3p overexpression significantly decreased IL-6 and IFNγ expression and increased IL-1β and cxcl10 expression in cultures of uninfected macrophages. Infected macrophages overexpressing miR-222-3p were characterized by increased NF-κB and IL-6 expression, as were infected macrophages without transfection. Another important finding was that miR-222-3p overexpression caused a small but significant increase in reactive nitrogen species production by infected macrophages, but did not affect their bacteriostatic activity against M. tuberculosis. Elucidating the functions of different microRNAs in regulating different pathogenic pathways in TB may lead to discovering new therapeutic targets. The detailed study of microRNAs that regulate immune-associated pathways will be useful for the design of miRNA mimetic molecules, either as inhibitors or as activators. Immune effects induced by miRNA drugs are currently a major challenge for miRNA therapeutics.
PD-1 AND Tim-3 Expression on different subpopulations of monocytes in chronic often recurrent herpesvirus infection
Authors: Rashchupkin Ivan M. , Meledina I. V. , Kotova M. A. , Zheltova O. I.
Published: July 2024

More than half of the world’s population is infected with the herpes simplex virus. In most cases, infection is not accompanied by symptoms, but in some people the disease occurs as a chronic infection with frequent and severe relapses. One of the most likely reasons for this may be a dysregulation of the immune system. In recent years, the role of checkpoint molecules, in particular PD-1 and Tim-3, in the regulation of the immune response and the functions of immunocompetent cells has been actively studied. Activation of PD-1 and Tim-3 on T cells has previously been shown to suppress the immune response. PD-1 and Tim-3 are also expressed on other immune cells, in particular monocytes. However, the expression of these molecules on monocytes during chronic viral infections has not been previously studied. The study was aimed at assessing the level of PD-1 and Tim-3 expression on various populations of monocytes in patients with chronic often recurrent herpesvirus infection. Twenty-six patients were recruited into the study. All patients received antiviral and immunomodulatory therapy in the immunological department. The number of classical, intermediate, and non-classical monocytes and the expression of PD-1 and Tim-3 on monocytes, were assessed by flow cytometry before and after the therapy. Monocytes were isolated from peripheral blood, and subpopulations were divided according to the level of expression of CD14 and CD16. In patients with herpes, a reduced number of monocytes was observed in comparison with healthy donors. The relative number of PD-1-positive monocytes, the mean fluorescence intensity of PD-1 and Tim-3, and the number of double-positive cells were reduced in herpes patients in all three monocyte subpopulations examined. Three months after therapy, the response to the therapy was assessed; patients who did not have a single recurrence of herpes within 3 months were considered to respond. Responding patients had a lower initial content of double-positive cells among intermediate and non-classical monocytes. The decrease in the level of PD-1 and Tim-3 positive monocytes during herpesvirus infection revealed in the present study may indicate the involvement of monocytes deficient in the expression of checkpoint molecules in the pathogenesis of the disease.
Post-COVID-19 syndrome: a discussion of onset timing
Authors: Berdiugina Olga V.
Published: July 2024

Introduction. Post-COVID syndrome (alternatively: chronic COVID syndrome, post-acute sequelae of COVID-19, long-haul COVID, long COVID, PASC, CCS) is a state of permanent or permanent presence of symptoms of a pathological process after COVID-19. Currently, there is no consensus on the timing of the onset of post-COVID-19 syndrome. The purpose of this study was to analyze the appearance of some post-COVID symptoms 1, 2 and 3 months after infection with the SARS-CoV-2 virus. Materials and methods. The analysis of clinical and laboratory data of 59 medical workers who had a mild or moderate form of COVID-19 was carried out. Three groups were formed: with the assessment of post-COVID-19 changes 1 month after the onset of the disease; with the detection of changes after 2 months; and with the detection of dysfunctions after 3 months. Results and discussion. It was found that 57.7% of respondents complained of fatigue, weakness, sleep disorders in the first month of observation. After 2 months, the frequency of complaints decreased by 42.3%, and by 3 months it increased again by 15.6%. A month after COVID-19, several cases were recorded when employees were unable to perform any, even very light, work. However, by 3 months of follow-up, 96.2% of the surveyed declared full recovery of efficiency. The appearance of muscle, joint, and spinal pain was reported more often in the first month after COVID-19, then the number of complaints decreased by half to 23–25% of the total number of observations. The assessment of the severity of the infection among the respondents of the three groups did not reveal significant differences between the data in 1, 2 and 3 months after COVID-19. Conclusion. An analysis of some post-COVID symptoms showed that in mild and moderate COVID-19, manifestations are observed 1, 2 and 3 months after infection with the SARS-CoV-2 virus. Multidirectional change dynamics were revealed, more often accompanied by a decrease in the manifestation of post-COVID syndrome 2 months after coronavirus infection, which may create a false impression of an absence of signs of post-COVID syndrome during this period.
Preparation of monoclonal antibodies for detection of recombinant flagellin C from Pseudomonas Aeruginosa
Authors: Zherebtsov Anton P. , Yakovleva I. V. , Gavrilova N. F. , Mikhailova N. A.
Published: July 2024

An important virulence factor in the pathogenesis of infections caused by Pseudomonas aeruginosa is flagellin: it serves as the main structural component of the bacterial flagellum and an acceptor for the TLR5 receptor of the innate immune system. Toll-like receptor 5 is able to bind bacterial flagellin and activate the anti-inflammatory transcription factor NF-kB through the adapter protein MyD88, which induces the production of anti-inflammatory cytokines. The inclusion of flagellin in recombinant proteins increased the ability to stimulate the production of anti-inflammatory cytokines and activate antigen-presenting cells. A number of experiments have shown that the use of flagellin as a molecular adjuvant in vaccines increases the expression of CD80, CD83, CD86 and MHC II molecules on the surface of dendritic cells, and also leads to an increase in the secretion of IFNγ and α-defensins by dendritic and NK cells; T cell proliferation and activation of antigen-specific cytotoxic T lymphocytes, as well as increased induction of antigen-specific IgG and IgA antibodies. Due to the natural and acquired resistance of P. aeruginosa to antibiotics, the available choice of antipseudomonas drugs is decreasing, and therefore the problem of developing effective therapeutic drugs to protect against this infection is of high medical and social importance. For this purpose, it seems promising to study the immunobiological properties of P. aeruginosa flagellin as a possible vaccine component. Based on this, in the Laboratory of Protective Antigens of the I. Mechnikov Research Institute of Vaccines and Sera, recombinant flagellin C (FliC) of P. aeruginosa was obtained, and its immunogenicity and protective properties were proven. However, the question of standardizing methods for screening and monitoring the resulting recombinant FliC protein remains open. To solve this issue, hybridomas producing monoclonal antibodies (mAb) of a given specificity were obtained, the basic immunochemical properties of mAbs were studied, and the possibility of using them as reagents in constructing a test system for identifying and standardizing the recombinant FliC protein upon its production was assessed. Purpose of the work: to obtain monoclonal antibodies to the recombinant flagellin C protein of P. aeruginosa; to study their basic immunochemical properties and to evaluate the possibility of using the recombinant FliC protein for screening and control.