Current issue
1 . 2022
Actual directions of modern immunology

Genetic constructs as adjuvants in vaccines based on adenoviral vectors

Abstract

The use of recombinant adenoviral (rAd) vectors for the production of vaccines against various diseases is an urgent task. Usually, rAd vaccines are highly immunogenic and do not require the use of adjuvants. However, in some cases, when antigen is secreted or cytoplasmic, need to increase the immunogenicity and protective properties of the vaccine. The introduction of molecular adjuvants in the composition of rAd vaccines as an auxiliary substance has shown conflicting results and has not found widespread use. A more promising approach is the introduction of adjuvants into the composition of rAd vaccines as genetic constructs. The review considers various genetic constructs that enhance the immunogenicity and protective properties of vaccines based on rAd vectors against various infectious diseases.

Vaccines and vaccination

Post-vaccination and post-infection humoral immune response to the SARS-CoV-2 infection

Abstract

Introduction. Mass vaccination of the population against SARS-CoV-2 in Russia and abroad has been going on for more than 2 years. Various types of vaccines are used for vaccination (vector vaccines, RNA vaccines, whole-virion vaccines). The first in the world was registered the Russian vector vaccine «Gam-COVID-Vac» («Sputnik V»), which is widely used in our country, as well as in many other countries of the world. An important area of research is the monitoring of the parameters of the vaccine-induced immune response their analysis in relation to the characteristics of the immune response caused by SARS-CoV-2 infection/COVID-19. Such studies are important both for assessing the intensity and duration of post-vaccination immunity, and for improving strategies for immunoprophylaxis and immunotherapy of coronavirus infection. The article presents the results of a study of the humoral immune response in patients with COVID-19 and vaccinated with «Sputnik V» in the period from autumn 2020 till the present time.

The aim of the work – a comparative study of the humoral immune response to SARS-CoV-2 infection and vaccination against COVID-19.

Material and methods. The content of antibodies against SARS-CoV-2 S- and N-proteins was studied in 449 blood serum samples of men and women (age 25–65 years). 5 groups of samples were formed: suffered from COVID-19 of mild and moderate severity, at different times after recovery (262 samples); immunized with the «Sputnik V» vaccine, at different times after injection of the 2nd component of «Sputnik V» (104 samples); suffered from COVID-19 of mild and moderate severity, and then vaccinated with «Sputnik V» (53 samples); vaccinated with «Sputnik V», and then suffered with COVID-19 (12 samples); revaccinated with the vaccine «Sputnik Light» (18 samples).

Results. To assess the content of IgG antibodies to S-protein and to N-protein of SARS-CoV-2 in human blood sera, an EIA diagnostic system was developed further certified by the Ministry of Health of Russian Federation. In people suffered COVID-19 in 90 % of cases the positive levels of IgG antibodies to SARS-CoV-2 S-protein persist 9 months after recovery, while the proportion of potential plasma donors and material for obtaining intravenous immunoglobulin with a high content of IgG antibodies to S-protein 1 month after the disease is 20 %. In 76 % of people vaccinated with «Sputnik V» high levels of IgG antibodies to S-protein are detected within 6 months after the course of vaccination. The content of IgG antibodies to S-protein in the blood sera of persons suffered COVID-19, and then 6 months after recovery vaccinated with the 1st component of the vaccine «Sputnik V» in 100 % of cases was high with a positivity coefficient above 8.1, regardless of the initial value, already on 7 and 21 days after injection. In persons vaccinated with «Sputnik V» and then passed COVID-19, a high content of S-specific IgG antibodies is observed in 100 % of cases. The examination of vaccinated persons who underwent revaccination with «Sputnik Light» showed a 100 % high level of antibodies against S-protein.

Conclusion. The developed test system may be suitable for assessing the content of IgG antibodies to SARS-CoV-2 antigens in COVID-19 patients, vaccinated and revaccinated. The performed study demonstrated that there is a intensive post-vaccination immunity for 6 months and post-infectious immunity for 9 months.

Immunogenetics

New mutations in the BTK gene in Russian patients with X-linked agammaglobulinemia

Abstract

Introduction. X-linked agammaglobulinemia (XLA) refers to primary immunodeficiencies (PIDs) with a defect in humoral immunity. This disease manifests mainly in patients with mutations in the BTK gene and is characterized by an inability to develop mature B-lymphocytes. Differentiating XLA from other PIDs with similar symptoms is essential for choosing the best treatment strategy as well as for genetic counseling of families, given the sex-linked inheritance of the disease. Currently, the most effective genetic screening approach is next-generation sequencing (NGS).

Aim – to confirm the diagnosis of XLA using a proprietary test system for preparing NGS libraries of BTK gene and characterize the clinical, immunological, and genetic traits of patients with XLA.

Material and methods. We developed a reagent kit to obtain a cDNA covering all 19 exons of the BTK gene and suitable for sequencing on the Ion PGM platform to confirm the diagnosis of XLA made according to ESID criteria. The study included 14 patients with unverified agammagolobulinemia from Russia from different families aged 9 to 40 years.

Results. BTK gene sequence analysis revealed 3 new putatively pathogenic mutations: 2 deletions NP_000052.1:p.Met570del/NM_000061.2:c.1708_1710delATG and p.Cys145Alafs*31/NM_000061.2:c.433delT, and 1 single-nucleotide polymorphism (SNP), NM_000061.2:c.1909-1G>A. 2 patients had no mutations in the BTK gene despite classic XLA symptoms. Most patients showed no significant association between genotype and phenotype, which is consistent with most of the literature.

Conclusion. This study confirms the effectiveness of NGS in assessing the mutational profile of the BTK gene and offers an accurate method for genetic confirmation of XLA diagnosis. Patients with an unconfirmed genetic diagnosis by genetic testing will be further investigated by other methods to identify autosomal mutations associated with the development of PID and to refine the diagnosis.

Cellular immunology

Analysis of the number and functional activity of T cells in AAA in mice lacking the IL-27 receptor

Abstract

Introduction. Abdominal aortic aneurysm (AАA) is a vascular disease of the abdominal aorta wall caused by the accumulation of large amounts of immune cells, inflammation and destruction of the vessel wall. The progressive growth of an aneurysm frequently leads to its rupture that can be fatal. Knowledge about the potential immune mechanism driving AAA development is still limited, impacting strategy for AAA treatment.

Aim of the study – to determine the role of the interleukin(IL)-27 receptor in the regulation of the number and functional activity of T cells in AАA in mice.

Material and methods. Apoe-/-Il27ra+/- and Apoe-/-Il27ra-/- mice were fed with high-carbohydrate/high fat diet (Western Diet, WD) and implanted with osmotic pumps filled with angiotensin II (Ang II) to induce the development of AAA. The number of T cells and their subpopulations in the suprarenal part of abdominal aorta/AAA tissue was investigated using flow cytometry and immunofluorescence analyses. The production of secreted cytokines was measured by ELISA in the supernatant of cells isolated from the suprarenal part of abdominal aorta/AAA. Two-tailed Student’s t-test was used to compare the 2 mouse groups (Apoe-/-Il27ra+/- and Apoe-/-Il27ra-/-).

Results. Suprarenal part of abdominal aorta/AAA of IL-27R deficient mice were characterized by limited number of T cells, in particular CD4+ and CD8+ subsets, along with reduced production of T-helper (Th) cytokines such as tumor necrosis factor(TNF)α and interferon-γ (IFN-γ) (Th1 cells), IL-4 and IL-13 (Th2 cells), IL-17A (Th17 cells).

Conclusion. Our data demonstrate that IL-27 receptor-mediated signaling affects the number and activation of T cells, suggesting that inhibition of this cytokine signaling pathway can be used to develop immunotherapy for the treatment of AAA.

Immunopathology

Content of pro- and anti-infl ammatory cytokines in the dynamics of experimental periodontitis in rats with chronic pain syndrome

Abstract

Introduction. The influence of chronic pain on the development of periodontitis is still unclear in spite of the wide spreading of periodontitis and chronic pain syndrome, especially in adult and elderly patients.

The aim of study – analysis of the influence of preliminary simulated chronic pain syndrome on the dynamics of pro- and anti-inflammatory cytokines in rats with experimental periodontitis development.

Material and methods. Chronic pain syndrome in Wistar rats was modeled by two-sided ligaturing sciatic nerves. Experimental periodontitis was modeled using the method supposed by A.I. Volozhin and S.I. Vinogradova. The following groups were formed in the study: naïve rats, animals with chronic pain syndrome, sham-operated rats, animals with experimental periodontitis, sham-operated rats with experimental periodontitis, animals with chronic pain syndrome and experimental periodontitis. The animals were deduced from the experiment on the 7th, 14th and 21st days after thread removing. The content of cytokines: IL-1β, IL-4, IL-6, IL-10, as well as TNFα and interferon-γ were detected in the blood plasm.

Results. The simulation of the chronic pain symdrome or sham operation didn’t cause the changes in the cytokines content in blood plasm. The increase of the concentration of pro- and anti-inflammatory cytokines in blood plasm of the rats with experimental periodontitis and sham-operated animals with experimental periodontitis was found throughout the experiment. The higher content of proinflammatory cytokines (IL-1β, IL-6, TNFα) in the blood plasma was revealed in the animals with experimental periodontitis and chronic pain syndrome throughout the experiment compared to the group, which experimental periodontitis was modeled. The concentration of anti-inflammatory interleukins in this group was less than one in rats with experimental periodontitis: IL-10 on the 7th–21st days and IL-4 on the 21st day. Indicated changes are conditioned by the influence of chronic pain syndrome on the development of experimental periodontitis in the animals.

Conclusion. The peculiarities of experimental periodontitis development in the rats with chronic pain syndrome were found in the study: the increase of the concentration of proinflammatory and decrease of the content of anti-inflammatory cytokines in blood plasm, compared to the group, which experimental periodontitis was modeled

Immunology of reproduction

Сontroversial role of innate immunity factors in impaired fertility in men affected by varicocele

Abstract

Introduction. The problem of male infertility caused by varicocele is currently of great medical and social significance, since this pathology is the most common reproduction disease among men, the frequency of which reaches 10–30 %, and deviations from the norm in spermogram parameters that occur against this background are observed in more than 40 % of patients. The study of the mechanisms of the innate immune system involved in the development of the pathological process in varicocele is an urgent task, and attracts the attention of an increasing number of scientists around the world.

The aim of this study was to examine the role of innate immunity factors in male infertility caused by varicocele.

Material and methods. The study included 60 ejaculate donors. The main group consisted of 27 patients diagnosed with varicocele. The comparison group consisted of 33 healthy donors. The obtained biomaterial was separated to fractions of spermatozoa and seminal fluid by density gradient centrifugation, nucleic acids were isolated from spermatozoa, and the expression level of TLR2, TLR4, HSP60, HSP70, HSP90, IL1B, IL18, and TNFA genes was determined by real-time polymerase chain reaction. The concentration of the HSP70 peptide in seminal fluid was determined by enzyme-linked immunosorbent assay, while the concentration of cytokines IL-1β, IL-18 and TNFα was determined by multiplex immunofluorescence analysis.

Results. In the course of a comprehensive analysis of the indicators of the innate immunity system in the spermatozoa and seminal fluid of patients with varicocele, we demonstrated: an increase in the expression levels of genes of recognizing receptors TLR2 and TLR4 in the spermatozoa of patients with varicocele by 6.9 times and 4.18 times, respectively; overexpression of heat shock protein genes HSP60, HSP70 and HSP90 – TLR2/4 ligands; an increase in the levels of expression of the genes of cytokines TNFA, IL1B, IL18 and the concentration of their protein products in seminal fluid in comparison with a group of healthy ejaculate donors. It has been demonstrated that an imbalance of innate immune factors in the ejaculate of men with varicocele correlates with impaired functional parameters of spermatozoa.

Conclusion. In this study, we revealed the ambiguous role of innate immunity factors in the ejaculate of men with varicocele. Toll-like receptors of spermatozoa are necessary for pathogen recognition and local immune protection, while heat shock proteins perform many functions that ensure spermatogenesis and fertilization. However, overexpression of these factors is associated with a decrease in both the fertile properties of spermatozoa and their number in the ejaculate. As a result of a comprehensive analysis of the innate immune system in the ejaculate, new diagnostic markers and targets for targeted therapy can be identified.

Clinical immunology

Dynamics of specific humoral response in COVID-19 patients

Abstract

Introduction. The pandemic caused by the SARS-CoV-2 virus is an important medical and social problem. It remains unclear the stability of the developed humoral immunity against the SARS-CoV-2 virus, the average duration of preservation of the titer of specific antibodies. The need to identify humoral mechanisms of immune defense in patients with COVID-19 determined the purpose of this study.

The aim of the study – to evaluate the dynamics of changes in the content of specific IgG antibodies against various antigens of the SARS-CoV-2 virus and B-lymphocyte subpopulations throughout the year in people who have had COVID-19.

Material and methods. The study included 90 patients who had undergone COVID-19 in various forms, subsequently divided into 2 groups: persons with asymptomatic and mild course (57 patients) and with moderate or severe course of the disease (33 patients). The dynamics of the concentration of specific antibodies to the SARS-CoV-2 virus was evaluated by enzyme immunoassay every 3 months for a year. The content of the total pool B-lymphocytes (naive B-lymphocytes, memory B-cells, regulatory B-lymphocytes) and various subpopulations was evaluated by flow cytofluorimetry.

Results. When assessing the dynamics of IgG to the S-protein of the SARS-CoV-2 virus, their preservation was noted by the 12th month after recovery. In patients with severe and moderate COVID-19 forms, these indicators are significantly higher. More severe forms of COVID-19 are accompanied by significantly higher content level of memory B cells throughout the observation period.

Conclusion. Moderate and severe forms of COVID-19 induce more persistent postinfectious humoral immunity, provided by an increase in memory B cells in comparison with lighter forms.

The content of platelet coaggregates with αβ-, γδ-Т-lymphocytes and their some minor subpopulations in the blood of healthy children

Abstract

Introduction. Platelets are able to enter into contact adhesive interactions with almost all blood cells. The formation of coaggregates of platelets and leukocytes in the bloodstream plays a certain role in the physiological processes of cell migration, and the degree of their interaction changes in pathology. In the literature, information on the adhesive interaction of platelets with leukocytes, and especially with lymphocytes and their subpopulations in the blood of healthy children is scarce.

The aim of the study – to research the relative and absolute content of coaggregates of platelets with lymphocytes, T-lymphocytes, αβ- and γδ-T-lymphocytes, their minor subpopulations – double negative and double positive T-lymphocytes (DNT and DPT cells) in the peripheral blood of healthy children of different age, as well as an assessment of the correlation between the studied parameters and the age of children.

Material and methods. The objects of the study were venous blood samples from 83 healthy children (boys and girls) aged from 7 months to 14 years. Monoclonal antibodies conjugated with various fluorochromes were used to detect lymphocyte-platelet coaggregates. Determination of the studied parameters was carried out using flow cytometry.

Results. The relative content and absolute amount of lymphocyte-platelet coaggregates in healthy children of different age groups have been investigated. A decrease in the absolute number of platelet coaggregates with T-lymphocytes, namely, with αβ-T-lymphocytes in the blood of older children, was established. At the same time, there was an increase in the relative content of the total pool of leukocyte-platelet coaggregates and coaggregates (platelets) with double negative T cells in older children compared with younger children.

Conclusion. The content of lymphocyte-platelet coaggregates depends on age. The absolute number of coaggregates of platelets with T-lymphocytes, αβ-T-lymphocytes decreases with age, and the content of coaggregates with γδ-T-lymphocytes remains unchanged. At the same time, there was an increase in the relative content of the total pool of leukocyte-platelet coaggregates and coaggregates (platelets) with double negative T cells in older children.

Differentiation of immunomodulatory effects of arginyl-alpha-aspartyl-lysyl-valyl-tyrosyl-arginine and glucosaminylmuramyldipeptide on effector functions and phenotype of functionally signifi cant subpopulations of neutrophilic granulocytes in an experimental model of viral-bacterial coinfection

Abstract

Introduction. The emergence of atypical infectious and inflammatory diseases, that are difficult to respond to standard therapy, is associated with the increasing frequency of manifestations of the combined pathology of coinfection and antibiotic resistance, which is a serious clinical problem. Clarification of the mechanisms of defective neutrophilic granulocytes (NG) functioning responsible for the emerging negative synergism of viruses and bacteria is urgent, and the assessment of the possibility of reorienting the functional potential of NG under the influence of immunotropic substances can allow creatingon of the new immunotherapeutic approaches for more effective treatment of viral-bacterial coinfections.

Aim of the study in model of viral-bacterial co-infection in vitro to assess the effect of arginyl-alpha-aspartyl-lysyl-valyl-tyrosyl-arginine and glucosaminylmuramyldipeptide on the effector functions of neutrophilic granulocytes (NG) and the phenotype of functionally significant subpopulations CD64CD32+CD16+CD11b+NG, CD64+CD32+CD16+CD11b+NG.

Material and methods. A study of 56 samples of peripheral blood (PВ) of apparently healthy adults was carried out. By sequential incubation of PВ with dsRNA, fMLP the experimental model of viral-bacterial co-infection was reproduced. A comparative assessment of the effect of hexapeptide arginyl-alpha-aspartyl-lysyl-valyl-tyrosyl-arginine (НP) and glucosaminylmuramyldipeptide (GMDP) on the phenotype and content of functionally significant subpopulations CD64CD32+CD16+CD11b+-NG, CD64+CD32+CD16+CD11b+-NG was carried out in the coinfections experimental model in an in vitro system; on activity NADPH oxidases of NG in NBT-test spontaneous and stimulated by Staphylococcus aureus (strain 209); on phagocytic function with S. aureus (strain 209): %FAN – % active phagocytic NG, % inactive NG (%ING), % NET – %NG formed NET, %NG in apoptosis.

Results. It was found that HP additionally activated the phenotype of the subpopulation previously activated in the experimental model СD64CD16midCD32brightCD11bbright-NG significantly reduced the number of NGs in the CD64+CD32+CD16+CD11b+-NG subpopulation, changing its phenotype to СD64brightCD16midCD32midCD11bmid-NG. GMDP did not affect the number of NGs in the CD64CD32+CD16+CD11b+ subpopulation, however, it caused a decrease in the expression density of all membrane receptors СD64CD16dimCD32dimCD11bdim-NG to the level of those in comparison group 1. In parallel, there was a decrease in the number of NGs in the activated subpopulation СD64+CD16+CD32+CD11b+-NGs, which was accompanied by a change in the phenotype in СD64midCD16dimCD32midCD11bmid-NGs.

Conclusion. The revealed differentiated effects of HP and GMDP effects on the content and phenotype of functionally significant NG subpopulations transformed in the experimental model of viral-bacterial coinfection can serve as a basis for the development of various immunotherapeutic strategies aimed at restoration of the normal NG function depending on the depth of the identified disorders.

Reviews

Immunoregulatory role of nucleated erythroid cells

Abstract

Nucleated erythroid cells (NEC) are the precursors of the most massive population of human cells – erythrocytes, for which functions of hemo- and immunoregulation have been shown at various stages of ontogenesis and in various organs and tissues of the human body. NEC perform this function by secreting cytokine proteins, growth factors, enzymes such as arginase-2, ROS, and by surface molecules PD-L1 and PD-L2. Their important regulatory role has been shown for the formation of fetoplacental immunosuppression, immunosuppression during pregnancy, suppression of the response against commensals in the gastrointestinal tract, in the pathogenesis of bacterial and viral infections in adults, in the pathogenesis of tumor growth and autoimmune diseases, as well as participation in the recognition of pathogen-associated molecular patterns using Toll-like receptors in fish and birds. Such qualities, together with their number and width of distribution, represent NEC as active participants in hemo- and immunoregulation, which makes it important to study their regulatory role in health and disease.

Jubilees

Professor Alexander Vasilievich Filatov

Abstract
Memorial dates

On the memory of Leonid Vasilyevich Kovalchuk: the creative way of a teacher and scientist (to the 85th birth anniversary)

Abstract

All articles in our journal are distributed under the Creative Commons Attribution 4.0 International License (CC BY 4.0 license)


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