Russian Journal of Infection and Immunity

The modern concept of international clinical trials (CT) has reached strict regulation in terms of norms and ethics and is convincingly reflected in a series of current legislative acts, at the global, regional and national levels. Of particular interest in this area are situations of dual loyalty related to moral, financial and legal gradation of research in countries with different levels in economic development and priorities for owning the investigational drug’s patent. Based on this, the new picture of the modern Russia geopolitical map on participation in international research requires adapting and revisiting the series of semantic characteristics. First of all, this concerns a fundamental change in Russia’s status that developed from a region for conducting research on foreign drugs to be further registered, to the position as the sponsor of research on domestic drugs in third countries, usually with limited economic and infrastructural resources. This metamorphosis is not only significant in terms of the qualitative redistribution of roles, but, in connection with the high rates of popularization and real increase in the volume of such research, for example, in Africa, it dictates a need to rethink the entire system of administrative and ethical approaches to their implementation. In this article, a targeted attention is first paid to the factor of special responsibility for the adoption and regulatory consolidation in the Russia the special ethical code for sponsor’s research in the context of their implementation in regions with limited resources. This task is coupled to introducing domestic experience as well as deep legal and information baggage accumulated by international practice into the protocol for the development of sponsor research in the Russia. The specificity is also of special importance because this work consists of the direct authorial involvement in the process for establishing and forming partnership ethical code based on the principles of justice, respect, care and honesty in research with unequal resources.

A variety of body tissues contain tissue-resident memory CD8+ T cells — long-lived multifunctional effector cells formed during the immune response to infection, which populate previously infected tissue long term. The localization of these cells is determined due to expression of a set of adhesive molecules that hold the cell in a specific tissue microenvironment, as well as the lack of molecules involved in exiting into the blood and lymphatic vessels. The program for establishing such cell phenotype can apparently be launched at various stages of T cell maturation during the immune response. Resident memory CD8+ T cells protect tissues from viruses and other intracellular parasites, kill transformed cells, and in some cases participate in the pathogenesis of immune-mediated inflammatory diseases. The accumulation of resident memory T cells can be induced by vaccination, and targeting these cells by vaccines, along with triggering a protective humoral response, appears desirable for the prevention of many intracellular infections. At the site of pathogen entry, resident memory CD8+ T cells can provide tissue protection quicker than circulating T cells, which is essential for preventing rapidly evolving viral infections. Localization of memory CD8+ T cells in tissues allows the pool of these cells to be increased without obvious restrictions. Accordingly, it is possible to carry out a large number of different vaccinations to stimulate these cells without losing the vaccines effectiveness. Finally, not all pathogens antigenic epitopes recognized by CD8+ T cells undergo rapid and systematic changes such as the surface B-cell epitopes of the same pathogens. Additional recruitment of resident memory CD8+ T cells in response to a vaccine may ensure greater coverage of pathogen variants and contribute to developing heterosubtypic immunity. The review presents data on resident memory CD8+ T cells in various tissues, their participation in the immune response to infections and vaccinations, as well as molecules that control their localization.

Preventive medicine holds significant promise for delaying and even preventing dangerous age-related pathologies. Advances in vaccine development, new preventive medications, cardiovascular implants and early diagnostic tools has increased life expectancy both in Russia and worldwide. Age-dependent genetic instability results in increased risk of developing a number of age-associated diseases. One consequence of this instability is the accumulation of senescent cells across tissues. Senescent cells lose functional activity, replacing normal cells, and are characterized by the secretion of proinflammatory cytokines, creating a chronic inflammatory background, which is the main characteristic of aging. Genetic instability is associated with higher activity of endogenous retroviruses, retrotransposons. Additionally, exogenic viruses and bacteria are able to damage cell genetic apparatus and induce disrupted transcription, translation and repair. The review discusses new approaches aimed at reducing the risk of age-related diseases by controlling genetic instability and viral infections, as well as mechanisms regulating genetic stability. Among the repercussions of disrupting such mechanisms are the development of proliferative, neurodegenerative diseases, cellular senescence and their contribution to chronic inflammation. Antiviral medications and compounds that suppress the activity of endogenous reverse transcriptases are proposed as promising preventive agents for suppressing age-related genetic instability. Although such substances are already used in the clinic to control human immunodeficiency virus, there are currently no medications suitable for long-term use without significant side effects. In this regard, it is primarily important to develop new generation medications that could be used as preventive agents and would meet efficacy and safety requirements, with minimal side effects. The review also explores other promising approaches such as senolytics (agents that selectively eliminate senescent cells) and senomorphics (substances that suppress proinflammatory in prevention of age-associated pathologies. Another perspective method for preventing genetic instability and accumulation of senescent cells is a search for highly specific targets and the development of immunization approaches that allow immune system to remove damaged and senescent cells with increased genetic instability. The proposed approaches can prolong health span and reduce the burden on healthcare system.

Human herpes virus 6B (HHV6B) is a ubiquitous causative agent of mononucleosis-like syndrome (MLS) and other diseases. The diversity of HHV6B infection may be due to virus genetic polymorphism. Molecular genetic diversity of circulating HHV6B, as well as its impact on the clinical and laboratory features of HHV6B infection have not been studied. The aim of the work was to study the intraspecific diversity of HHV6B circulating among children in Nizhny Novgorod (Russia) region, and to investigate its effect on MLS clinical and laboratory features. Blood leukocytes and saliva DNA samples from healthy children and children with HHV6B infection were analyzed for HHV6B DNA detection. Original intraspecific HHV6B classification based on detecting signature nucleotide substitutions in the sequence of the U90 gene fragment, was used for virus typing. A relationship between HHV6B genovariant and clinical/laboratory MLS characteristics was studied using multivariate statistical methods. Circulation of four HHV6B genovariants was shown among children in Nizhny Novgorod region: HHV6B/GV2e, HHV6B/GV2b (dominant genovariants), HHV6B/GV1a and HHV6B/GV2a (minor genovariants). Children with MLS infected with HHV6B/GV2e genovariant were more likely to have laboratory signs of cytolysis and increased peripheral blood lymphocyte count. Children with MLS infected with the HHV6B/GV2b genovariant were more likely to have higher mature neutrophil level. An increase of HHV6B viral load in leukocytes was accompanied by aggravated intoxication symptoms. Co-infection with the Epstein–Barr virus (EBV) led to more severe symptoms of acute tonsillitis, hepatosplenomegaly, and lymphadenopathy. Patients with EBV were more likely to have higher ALAT and lymphocyte level along with decreased mature neutrophil count. In children with MLS, EBV coinfection caused smaller HHV6B viral load in blood leukocytes (in children with HHV6B DNA 10 or more copies/105 cells) and lower HHV6B DNA detection frequency in saliva (in children with HHV6B DNA less than 10 copies/105 leukocytes). The results show the influence of genetic HHV6B polymorphism upon the clinical and laboratory features of MLS in children, and also demonstrate that HHV6B/GV2e and EBV cause similar disease symptoms.

In recent years, the role of former saprophyte bacteria in the etiology of human infectious diseases has become an increasingly pressing issue. Of particular interest are non-fermenting gram-negative bacteria, many of which are members of the rhizobiome in some plants, soil and water microbiome. Increasingly, scientific literature covers cases of infections caused by members of the order Flavobacteriales, namely the genus Chryseobacterium, Elizabethkingia and Empedobacter of the Weeksellaceae family. Microorganisms of the latter are causative agents in several human diseases, including urinary tract and respiratory tract infections. The aim of our study was to investigate the biological properties of bacteria of the genus Chryseobacterium, Elizabethkingia and Empedobacter of the Weeksellaceae family isolated from patients with cystic fibrosis and to assess their pathogenetic potential. To analyze the biological properties and pathogenetic potential, 32 clinical strains of bacteria belonging to the Weeksellaceae family, isolated from biological material samples of the respiratory tract from cystic fibrosis patients, were selected for the study. Differential diagnostic media and plate test systems were used. DNA was isolated using 5X ScreenMix (ZAO Evrogen) and PCR with gel electrophoresis was performed. Bacteria of the Weeksellaceae family are characterized by a wide range of enzymes, especially C. arthrosphaerae and E. meningoseptica. It was found that strains of the Chryseobacterium, Elizabethkingia and Empedobacter genus are carriers of virulence genes, exhibit pathogenicity and participate in chronic respiratory infections in patients with cystic fibrosis. Phenotypic methods for studying enzymatic activity showed that all the examined strains of bacteria of the Weeksellaceae family are characterized by gelatinase activity, and representatives of the genus Chryseobacterium spp. — also by the ability to synthesize plasma coagulase. The potential to produce other enzymes of aggression is less pronounced and was strain specific. Analysis of the genetic profile of virulence factors in the studied bacteria showed that in their genome, genes encoding the enzymes lecithinase, hyaluronidase, elastase and hemolysin were most often found, and in bacteria of the genus Chryseobacterium spp. and Empedobacter spp. — neuroaminidase, which contribute to overcoming tissue barriers, pathogen invasion and spread throughout host tissues. The obtained results and scientific literature data indicate a special clinical role for bacteria of the Weeksellaceae family, which determines the importance of a deep study of the biological properties and virulence factors in members of the genus Chryseobacterium spp., Elizabethkingia spp. and Empedobacter spp.

Hepatitis C virus (HCV) infection remains a serious global healthcare challenge, often leading to chronic disease with complications such as liver cirrhosis, hepatocellular carcinoma, and extrahepatic manifestations. According to the World Health Organization (WHO), an estimated about 50 million people lived with chronic HCV infection in 2022, highlighting the ongoing need to understand the immune mechanisms driving disease progression. HCV may evade of immunosurveillance, so that disease progression is linked to specific immune response, whereas reactivation risk after antiviral therapy exists, which together account for a need to gain understanding of underlying immune mechanisms. HCV may infect lymphocytes primarily B-cells serving as a virus reservoir and result in diverse systemic complications. The current study focused on analyzing peripheral blood CXCR3+ B-cells at various differentiation stages in patients with chronic HCV infection to assess a potential link to clinical and laboratory markers of disease progression. Blood samples collected from 58 patients with chronic HCV infection and 27 healthy controls were examined. When analyzing flow cytometry data, we noted a significant B-cell redistribution in HCV infection compared to control samples presented as a shift towards activated mature, resting memory and ‘double negative’ B-cells characterized by increased expression of chemokine receptor CXCR3. Flow cytometry analysis revealed no significant difference in total B-cell (CD19+) but elevated memory B-cells (CD27+CD19+) (p = 0.037). CXCR3 expression peaked on memory B-cells and increased across all B-cell subsets in HCV patients (p < 0.001). Redistribution toward differentiated B-cell subsets — double-negative (CD38–CD27–), resting memory (CD38–CD27+), and activated mature (CD38+CD27+) B-cells was observed, with elevated CXCR3+ percentage in the latter two subsets (p = 0.017 and p = 0.001, respectively). HCV viral load correlated positively with CD38+ B-cells and CXCR3+ naïve/activated mature subset counts but inversely with CD38+ B1/B2/memory cell levels. Genotype 1 and advanced fibrosis (F3/cirrhosis) were associated with reduced B2 cells and increased CXCR3+ B1/B2 subset levels. These findings suggest that chronic HCV infection drives B-cell differentiation and CXCR3-mediated recruitment to the liver, implicating CXCR3 in disease progression.

Infectious agents have closely interacted with the human immune system, acquiring a set of highly sophisticated mechanisms for modulating immunity. One of the survival strategies for viruses, bacteria, protozoa, helminths and fungi is to target the regulatory T cell network (Treg: CD4+CD25hiCD127–) that controls immunopathogenic responses in many infections. Not only pathogens but also commensals are able to directly induce the conversion of naive T cells into suppressive Foxp3-expressing Tregs, while others activate pre-existing natural Tregs, in both cases suppressing pathogen-specific effector responses. However, Tregs can also contribute to immunity under certain conditions, such as at the initial stages of infection when effector cells must gain access to the site of infection, and subsequently in ensuring the generation of effector memory cells. It is noteworthy that currently little information on whether infections selectively drive pathogen-specific Tregs, and if so, whether such cells are also reactive to autoantigens are available. Further analysis of Treg subset specificity, along with a clearer picture of relative dynamics during the disease, should lead to rational strategies of immune intervention to optimize immunity and eliminate the infectious process. Thus, restoration of Treg function is important in the treatment of infectious, autoimmune and other diseases and can serve as a marker of their successful treatment. The article assesses the effect of exometabolites of derived from permafrost Bacillus bacteria obtained at different temperature conditions of their cultivation on the activity of Treg and effector T lymphocyte differentiation. Significant differences were established: secondary microbial exometabolites affect Treg (CD4+CD25hiCD127–) differentiation and expression of activation markers (CD69, CD25, HLA-DR) on CD4+ and CD8+ T lymphocytes. This effect is regulated by the type of metabolites obtained at different temperatures — “cold” (obtained at 5°C of bacterial incubation), “medium-temperature” (at 22°C) and “heat” (at 37°C) metabolites. In this case, an increase in the Treg level is associated with lower differentiation activity of CD4+ T-lymphocytes exposed to “cold” secondary exometabolites, a decrease in the differentiation activity of CD8+ T-lymphocytes treated with “warm” secondary exometabolites, and a roughly equivalent effect on the differentiation activity of CD4+ and CD8+ T-lymphocytes acted upon by “medium-temperature” secondary exometabolites.

The article is devoted to the development of a method for detection of viral RNA of two highly pathogenic zoonotic viruses from the genus Henipavirus — Hendra and Nipah using real-time reverse transcription polymerase chain reaction. In the natural environment, these viruses are carried by flying foxes in the family Pteropodidae. Horses and pigs, respectively, are susceptible to infection. The diseases are also transmitted to humans through contact with sick animals, their biological excreta and from person to person. In infected humans and animals, clinical signs of infection may be asymptomatic, or may present with flu-like symptoms at the onset of the disease and progress to neurologic disease and acute respiratory infection, followed by death. In Australia, the subunit vaccine HeV-sG is used against Hendra virus in horses. There is no treatment or vaccine for Hendra or Nipah viruses for humans. The need to develop new detection methods and search for new viral targets remains an urgent task due to the large area of distribution of the described viruses, high contagiousness and mortality of animals and humans. The study describes the original designed primers and probes for conserved regions of the genomes of two viruses: the gene encoding the nucleocapsid protein of Hendra virus and the gene encoding the glycoprotein of Nipah virus. Synthetic controls for the extraction and reverse transcription PCR stages have been created, confirming the quality of the developed method. Biological samples from healthy people (blood plasma, swabs from oral and nasopharyngeal mucous membranes, cerebrospinal fluid) with the addition of artificial controls passed the stages of sample extraction and real-time reverse transcription PCR, thus confirming the quality of control samples. The detection limit of the described viral RNA identification methods was determined as 100 copies/mL for Hendra virus and 1000 copies/mL for Nipah virus. The amplification transit time is less than 90 minutes. The developed method will help in epidemiologic control of the spread of these infections, can be used in the diagnosis of Hendra and Nipah viruses and for solving research tasks to study the properties of these pathogens.

Patients with COVID-19 are characterized by markedly elevated levels of cell-free DNA in the peripheral blood, and recently neutrophil extracellular trap DNA strands were detected in the blood plasma of individuals with post-COVID syndrome. Purpose of the study — to develop a methodological approach (technology) for detecting cell-free DNA and to assess its level in the blood of individuals with post-COVID syndrome. Materials and methods. The study enrolled patients with post-COVID syndrome who had severe COVID-19 (experimental group, n = 8, aged 53 to 64 years); patients with severe COVID-19 (comparison group I, n = 5, aged 48 to 67 years); apparently healthy volunteers of different age groups: 18–30 years old (comparison group II, n = 12) and over 60 years old (comparison group III, n = 10, over 60 years old), in whose blood there were no specific IgG antibodies to the SARS-CoV-2 virus. The study was performed in microvolume whole blood samples. Results. To determine the content of intracellular DNA fragments in the blood, a flow cytofluorimetric analysis method was developed based on adding a propidium iodide dye solution to the blood together with the CD45-FITC marker used in leukocyte immunophenotyping according to the Lyse/No-Wash protocol to differentiate intact cells from cellular debris. In healthy subjects, after adding a reagent that lyses erythrocytes and fixes leukocytes to the blood, the dye penetrates into the cells and stains only the intracellular DNA in intact diploid leukocytes. The proportion of weak DNA fluorescence signals from extracellular DNA fragments in the total number of pulses recorded by the cytometer in this case not exceeds 3.2 (2.0–5.6)%. With age in case of the disease, this pattern increases to an average of 13.4 (10.1–18.6)%. In individuals with severe COVID-19, very high magnitude of the studied parameter was recorded — at the level of 82 (68.0–88.6)%. In COVID-19 convalescent patients, high levels of extracellular DNA remained for a month, gradually decreasing by the month 3 to 40.5 (27.4–52.0)%. Conclusion. According to the data obtained, the determination of extracellular DNA using the developed technology allows us to characterize the severity of COVID-19 and assess the compensatory capabilities of the body in the post-COVID period.

Among the FOXO (Forkhead box O) family proteins, which are the most studied and widely represented in various human organs and tissues, transcription factors FOXO1 and FOXO3 are of special importance. Numerous studies evidence about their crucial role in maintaining cell metabolism and homeostasis, as well as in the pathogenesis of various pathological conditions. The involvement of the FOXO-signaling pathway in the pathogenesis of pulmonary sarcoidosis is also confirmed by RNA sequencing data. FOXO activation is related to the regulation of autophagy processes, the disruption of which is noted in pulmonary sarcoidosis, a systemic immunoinflammatory disease of unknown etiology, characterized by the formation of epithelioid cell granulomas in various organs, primarily in the lungs. The study was aimed at investigating expression of FOXO1, FOXO3 and BECLN1 genes in peripheral blood leukocytes from patients with chronic pulmonary sarcoidosis compared with apparently healthy donors (control). It is shown that the expression of transcription factors FOXO1 and FOXO3 genes is significantly higher in peripheral blood leukocytes (PBL) from patients with pulmonary sarcoidosis stage II, with chronic course (stabilization of the condition), in the absence of therapy compared to the control (p < 0.01 and p < 0.001). The PBL BECN1 gene mRNA level from patients with chronic pulmonary sarcoidosis is also higher compared to control group. Correlation analysis revealed a close positive relation between the expression of FOXO1, FOXO3 and BECN1 genes, with Spearman rank correlation coefficients comprising 0.69 (FOXO1/BECN1) and 0.61 (FOXO3/BECN1) (p = 0.0002 and p = 0.0016, respectively). Thus, in stage II pulmonary sarcoidosis patients without therapy, FOXO1/3 activation is likely associated with upregulated BECN1 gene expression encoding the autophagy protein BECLIN1 (ATG6). Numerous molecular genetic markers that contribute to the risk of pulmonary sarcoidosis have been identified. Studying the underlying molecular mechanisms is essential to better understand the disease pathogenesis not only allowing to gain insight into the clinical symptomatology but also to predict the disease outcome. In addition, increased knowledge on pathogenesis contributes to developing more targeted, effective and safe treatment methods for pulmonary sarcoidosis, taking into account individual characteristics of each patient’s health status.

The new COVID FLiRT vs. previous variant does not pose a greater danger; on the contrary, the new strain has a lower pathogenicity, although it is highly contagious. The article describes a rare case of pulmonary cryptococcosis in a patient with Wiskott-Aldrich syndrome co-infected with the FLiRT strain. Case description. Patient G.P., 31 years old, was in hospital for several hours. He was admitted for emergency indications with complaints of inhale chest pain, dry cough, and body temperature increased to 38°C. It is known from the medical history that the patient had been ill for about two weeks. In the last few days, before hospitalization, he noted a rise in body temperature to 37.3–38.1°C, and his cough worsened. In this regard, he called an ambulance and was taken to the emergency room for diagnosis and treatment. In childhood, he was diagnosed with Wiskott-Aldrich syndrome. In recent years, he presented no complaints from the internal organs. According to the epidemiological history, it was revealed that the patient did not travel to endemic regions 2 weeks before the onset of the disease, nor contact with people suspected of being infected with SARS-CoV-2, or patients whose diagnosis was confirmed by laboratory tests. A general blood test revealed leukocytosis, lymphopenia, an increase in procalcitonin to 4 ng/ml. Computed tomography of the chest organs revealed signs of interstitial pneumonia and small foci (2–7 mm) in the surrounding lung parenchyma. Despite the treatment started, a cough with discharged pinkish sputum suddenly began and death occurred. Autopsy showed alternation of moderately airy alveoli with focal emphysematous alveoli. In the lumen of the alveoli, leukocyte infiltration, edematous fluid, foci of granulomas with cryptococci, lymphohistiocytes, macrophages and single multinucleated giant cells were determined, on the interalveolar septa. Conclusion. Based on the pathological examination and the description of one of the presented cases, it can be assumed that the new COVID FLiRT variant in combination with Cryptococcus neoformans can lead to a severe form of the disease.