A synthetic peptide mimicking the antigenic site of F protein suppresses of respiratory syncytial virus infection in vitro
Introduction. Respiratory syncytial virus (RSV) is one of the most common pathogens causing respiratory diseases. There are no effective vaccines against RSV infection. At the same time, the use of monoclonal antibodies for immunoprophylaxis is limited by its high cost. Ribavirin is used for pharmacotherapy, but it causes numerous side effects. One of the most promising approaches is the use of natural and synthetic peptides as antivirals. Detailed studies of virion structure identified leading role of viral glycoprotein F in initiation of the infection. Molecular and immunological mapping of the F protein revealed the functional HR-1 and HR-2 domains. The flexibility of these domains provides the fusion of the virion with the cell. At the same time, inside HR-1 domain was identified so-called zero antigenic site Ø. Inactivation of this site significantly inhibits the infectious process.
The aim of the study was to design a peptide that mimics the antigenic site Ø of the F protein and its branched analog and study the antiviral properties of these peptides against RSV.
Material and methods. The peptides were synthesized by the solid phase method, and the high-performance liquid chromatography was used for purification. Authenticity was confirmed by mass spectrometry. Cytotoxicity was studied in the MTT test. Antiviral activity was studied in in vitro experiments using MA-104 cell culture, which in sensitive to RSV. The ELISA was used to study the ability of peptides to interact with RSV and with the cell receptor.
Results. Linear peptide KK-44 interacted with the virus, but had no significant effect on the infectious process. At the same time, the branched analog (KK-45 peptide) significantly inhibited RSV infection in vitro using several possible mechanisms: 1) by interacting with the F fusion protein, preventing its functioning; 2) by competitive inhibition of the receptor – nucleolin protein.
Conclusion. The branched peptide KK-45 possesses antiviral properties against RSV, which makes it promising for possible therapeutic applications.
Generation and characterization of stable antigen-specific T-regulatory cells using blockers of cyclin-dependent protein kinases
Introduction. In the immune system, regulatory CD4+CD25highFoxP3+T cells (Treg) are important players in tolerance induction. Treg suppress inflammatory reactions of various etiologies, including autoimmune and transplant reactions. Using the inherent regulatory potential of Treg induced in vitro, it is possible to achieve suppression of inflammatory responses to a specific antigen without affecting the immune response as a whole.
Aim of the study – obtaining antigen-specific FoxP3+Treg with characteristic immunosuppressive properties in mixed lymphocyte culture.
Material and methods. Object of this study were male mice of C57BL/6 and Balb/c lines. The resulting dendritic cells from C57BL/6 mice and CD4+ splenocytes from Balb/c mice were co-cultured to obtain antigen-specific CD4+ T cells. After that, CD4+ T cells were cultured with AS2863619, TGFβ and IL-2 to transdifferentiate antigen-specific effector T lymphocytes to antigen-specific CD4+CD25highFoxP3+Treg. The phenotype of the resulting Treg was assessed using flow cytometry. The tolerogenic potential of Treg was assessed using a mixed lymphocyte culture.
Results. It was shown that the combination of anti-CD3 and AS2863619 leads to a 10-fold increase in the relative number of CD25highFoxP3+Tregs in a culture of CD4+ T lymphocytes compared to unstimulated lymphocytes. Flow cytometrшс analysis showed that the population of effector memory T cells predominate in the antigen-specific Treg culture. At the same time, in the culture of non-antigen-specific Treg, only about half of the subpopulations consisted of resident memory T cells. In a mixed culture of lymphocytes, it was found that in the presence of non-Ag-Treg the proliferation of Balb/c CD4+ T lymphocytes increased by 2.5 times in response to the addition of the C57Bl/6 antigen. However, in the presence of Ag-Treg, even with the addition of C57Bl/6 antigens, the level of proliferation of Balb/c CD4+ T lymphocytes does not differ from the control group.
Conclusion. The use of AS2863619 in the cultivation of antigen-specific CD4+ lymphocytes enhances the generation of Ag-Treg. At the same time, the resulting Ag-Tregs have the phenotypic properties of effector memory Treg and functionally suppress the proliferation of CD4+ lymphocytes against antigens, which makes it reasonable to study their ability to induce immunological tolerance and suppress the immune response.
Dynamic assessment of the intensity of the immune response to SARS-CoV-2 infection and immunization against COVID-19 with the vaccine «Sputnik V»
Introduction. Searching for the best solution of preventing the development of complications after SARS-CoV-2-infection and reducing mortality from this highly contagious disease, is significantly complicated by the high virulence and constant mutations of the virus. Dynamic monitoring of the immune response of the population is still an important part in improving the algorithms for diagnosing and treating COVID-19. This work continues the series of studies on the features of SARS-CoV-2-specific immunity in convalescents and recipients of the vaccine «Sputnik V».
The aim of the study – dynamic comparative assessment of the intensity of the immune response to SARS-CoV-2 infection and immunization against COVID-19 with the vaccine «Sputnik V» after 4 and 13 months after vaccination.
Material and methods. The content of IgG-antibodies to the S- and N-antigen of SARS-CoV-2 was studied in 74 samples of blood sera of individuals aged 18 to 70 who recovered from COVID-19 and then underwent a full course of immunization with the vaccine «Sputnik V» after 4 and 13 months. We also studied 12 samples of blood sera from individuals who’d completed a full course of immunization with the vaccine «Sputnik V» and did not suffer from COVID-19. Ranking by age or gender was unnecessary.
Results. A high level of IgG-antibodies to the SARS-CoV-2 S-protein is observed in 85 % of those who have been recovered from COVID-19 after 4 months after vaccination and in 67 % after 13 months. A significant increase in the proportion of high levels of IgG-antibodies to the nucleocapsid protein with an almost twofold decrease in the proportion of the «grey zone» and negative results was shown, possibly due to of a contact with the SARS-CoV-2 strain «omicron» or suffering from COVID-19asymptomatically.
Conclusion. The study shown the presence of intense post-vaccination and post-infection humoral immunity response in people, who had previously recovered from COVID-19, after 4 and 13 months after vaccination.
Effect of capsular polysaccharide and detoxified lipopolysaccharide Shigella sonnei on tumor node growth and cytokine synthesis in C57Bl/6 mice after inoculation of melanoma B16
Introduction. Melanoma represents an immunogenic tumor which cells are recognized by the immune system. Under certain conditions the immune system can suppress the development of malignant neoplasm, as evidenced by clinical observations of spontaneous regression and complete elimination of the tumor in some patients.
One of the possible approaches to the induction of regression of malignant tumors is the stimulating the cells of the immune system with products of bacterial origin. In this stimulation Toll-like receptors play a key role, recognizing the conservative structures of microorganisms and activating the innate and adaptive immune response.
Aim of the study – to investigate the ability of the two bacterial compounds: detoxified lipopolysaccharide (Ас3-S-LPS) and capsular polysaccharide (CPS) of Shigella sonnei phase 1 to stimulate antitumor immune response in experimental animals with transplanted malignant melanoma.
Material and methods. The transplanted melanoma cell line B16 was used as a model for the study. The B16 melanoma cell line was provided by the Cellular Immunity Laboratory, Research Institute of Experimental Diagnostics and Tumor Therapy (NII EDiTO), N.N. Blokhin NMRCO, MOH of Russia. The antitumor activity of the drugs was estimated by the effect on the growth of primary tumor nodes in mice C57Bl/6J after introduction of tumor cells. When studying the mechanisms of antitumor activity, the effect of drugs on the formation of nitric oxide and the synthesis of cytokines (IL-1β, IL-6, IL-10, TNF and GM-CSF) was assessed.
Results. It has been shown that the administration of Ac3-S-LPS and CPS to mice leads to a slowdown in the growth of primary tumor nodes (PTN), which is not associated with a direct cytotoxic effect on tumor cells. Ac3-S-LPS and CPS, as well as Shigella sonnei native LPS, are unable to induce nitric oxide synthesis by B16 melanoma cells. However Ac3-S-LPS and native LPS induce nitric oxide synthesis in PTN cell culture. We have shown that B16 melanoma cells when cultured in vitro spontaneously produce three of five studied cytokines (IL-1β, IL-6 and IL-10) and do not synthesize TNF and GM-CSF. The addition of Shigella sonnei LPS or Ac3-S-LPS to cells stimulated the synthesis of IL-1β, IL-6, IL-10 and did not affect the synthesis of the other two cytokines.
The cytokines TNF, GM-CSF, and IL-4 were detected in the supernatants of PTN cultures. Their concentration increased when Ac3-S-LPS was added to cultures. The introduction of the Ac3-S-LPS preparation directly into the PTN led to the stimulation of IL-10 synthesis and did not affect the synthesis of other cytokines.
Conclusion. The antitumor activity of 2 bacterial compounds was studied on the model of transplanted melanoma: Ac3-S-LPS and CPS of Shigella sonnei phase 1 microorganisms, which have not been previously studied in this aspect. The obtained results allow us to classify them as TLRs agonists that suppress the growth of B16 malignant melanoma tumor after inoculation in experimental animals as a result of stimulation of innate immune responses. The study of the effect of cytokines induced by the interaction of bacterial ligands with TLRs on immune system and tumor cells leads to a deeper understanding of the pathophysiology of carcinogenesis and may contribute to the development of targeted therapy techniques for inhibiting tumor growth.
Prognostic significance of phenotypic changes of lymphocytes in hemorrhagic fever with renal syndrome
Introduction. Hantaviruses are widespread in nature, affecting animals and forming natural reservoirs. In people living in natural foci, these viruses most often cause hemorrhagic fever with renal syndrome (HFRS) – a serious disease with vascular and kidney damage with the likelihood of death, which has medical and social significance.
Aims – immunopathogenic substantiation of the role of certain lymphocyte phenotypes in the development of severe HFRS and the development of criteria for predicting the severity of the course in the initial period of the disease.
Material and methods. 37 people were examined, among whom 24 people were diagnosed with HFRS (16 patients of moderate course and 8 patients of severe course). The remaining 15 people were clinically healthy and made up the comparison group. All persons in the first days of the disease were examined by flow cytometry of the blood сells with the determination of lymphocyte phenotypes in the innate and adaptive immunity.
Results. It was found that two types of immunological shifts, depending on the HFRS severity, were observed. In the severe course of the disease, there was a decrease in the ratio of T-helper/CTL (CD3+CD4+/CD3+CD8+), as well as an increase in the ratio of NKG2D+ CTL/CD8+ Treg (CD3+CD8+CD314+/CD3+CD8+FoxP3+). The ratio of NKG2D+ CTL/CD8+ Treg was more diagnostically significant and was called the cytotoxic-regulatory index (CRI). If the patients in HFRS had CRI > 2.4, it was recommended to be attributed them to the risk group for the development of a severe course of the disease.
Conclusion. The results create a scientific and clinically significant basis for predicting severe HFRS using the ratio of NKG2D+ CTL and CD8+ Treg that should be clarified further in a larger group of patients.
Dynamics of clinical and immunological parameters in patients underwent a moderate COVID-19 and received therapy with janus kinase inhibitor
Introduction. The study of the pathogenesis of COVID-19 remains one of the main tasks of modern science. Having studied the mechanisms of the development of the disease, it is possible to reduce the risks of developing a severe course of the disease much more effectively. Respiratory failure, «cytokine storm», thrombotic complications – all this is associated with an unfavorable prognosis of a new coronavirus infection. The targeted therapy used to combat the development of these complications has been successful in treating patients with COVID-19. One of the proactive mechanisms in the pathogenesis of the «cytokine storm» is the blockage of the JAK-STAT signaling pathway with the janus kinase inhibitors.
It is very important to represent the effectiveness of the drugs used, as well as its safety, not only in the acute period of the disease, but also in the post-COVID period. The area of interest are changes of clinical and laboratory parameters and indicators of innate and adaptive immunity after the disease in patients receiving therapy with janus kinase inhibitor.
The aim was to study the clinical and immunological parameters of patients underwent moderate COVID-19 and received therapy with janus kinase inhibitor in the acute period of the disease and 6 months after discharge/recovery.
Material and methods. 30 patients hospitalized with a diagnosis of «coronavirus infection, virus identified, COVID-19, moderate form; complication: interstitial pneumonia» were examined. A janus kinase inhibitor (baricitinib) was included in the complex treatment of patients in this group. The comparison group consisted of 20 patients hospitalized with an identical diagnosis, in the treatment of which the janus kinase inhibitor was not used. The dynamics of laboratory parameters (general clinical, biochemical and immunological) were evaluated at the background of therapy with janus kinase inhibitors upon admission to the hospital, upon discharge as well as 6 months later.
Results. The effectiveness of the use of the janus kinase inhibitor baricitinib is manifested in a decrease of the level of markers for the prognosis of severe infection, as well as reduction of the hospitalization time of patients. By the time of discharge /recovery, there is a decrease in the levels of C-reactive protein, lactate, fibrinogen. The level of lactate dehydrogenase by the time of discharge / recovery is reduced to reference values in the group with the use of baricitinib. 6 months after discharge from the hospital, the normalization of the level of C-reactive protein to reference values is noted. By the time of discharge, a decrease in the levels of IL-10, IL-6 and IFN-γ was recorded. Regardless of the use of the janus kinase inhibitor, changes of the innate and adaptive immunity parameters persist as the increased content of circulating immune complexes and NK cells (CD16+). In case of the use of dexamethasone in preventive therapy without the use of baricitinib in patients 6 months after discharge from the hospital, the level of CD3+CD4+ lymphocytes was significantly higher, and the level of serum IgG was lower.
Conclusion. The use of baricitinib in complex treatment, including GCS therapy, antiviral therapy, anticoagulants, leads to a significant decrease of parameters indicating the development of a «cytokine storm», as well as to decrease in the cytopathic effect of the virus. Dysregulatory processes in the immune system, that occur during the disease, persist even 6 months after the recovery of patients underwent moderate COVID-19.
The type of cytokines production that regulate NK activity in women with uterine leiomyoma
Introduction. Natural killer cells (NK) play an important role in the regulation of tumor growth. The control of NK activity is largely carried out by the microenvironment, in which cytokines play the main role.
The aim of the study was to evaluate the effect of IL–18, IL-27 and IL-35 regulating NK function in women with uterine leiomyoma (LMM).
Material and methods. The study involved 51 womens of reproductive age with symptomatic intramural LMM and 12 practically healthy women without this pathology. The level of cytokines IL-18, IL-27 and IL-35 in peripheral blood serum, endometrial tissue and myomatous nodes lysates was assessed by enzyme immunoassay.
Results. In women with LMM, the serum IL-18 level was lower compared to women without LMM. IL-27 in serum was detected only in 20/51 (39.2%) women with LMM. Women of reproductive age with LMM were characterized by a higher level of IL-27 in endometrial tissues than women without LMM. In the tissues of myomatous nodes, lower levels of all studied cytokines regulating the activity of NK were detected in comparison with those in the endometrium.
Conclusion. The presence of myomatous nodes affects the production of cytokines regulating the activation of NK in endometrial tissues, however, in the tissues of the myomatous nodes themselves, the production of cytokines, both with activating and inhibitory activity involved in the regulation of NK function, remains relatively low.
The effectiveness of interferon gamma inclusion in the tuberculosis chemotherapy: experimental study
Introduction. Currently, in phthisiology there is a need for new drugs and medicines with new mechanisms of action that could be effective against sensitive and resistant forms of tuberculosis, as well as prevent the development of mycobacterium resistance to anti-tuberculosis drugs, reduce the duration of treatment and the financial burden of this nosology.
Aim of the study is the evaluation of the therapeutic effect of interferon gamma adding to Isoniazid therapy of tuberculosis in guinea pigs caused by the M. tuberculosis H37Rv strain sensitive to all anti-tuberculosis medicines.
Material and methods. We evaluated the effectiveness of treatment by such indicators as animal life expectancy, weight gain, the presence and intensity of bacterial excretion by microscopy and culture, as well as the nature and severity of histological changes in internal organs.
Results. As a result of studying the therapeutic effect of the drug Ingaron (human recombinant interferon gamma) in infection caused by M. tuberculosis H37Rv, we noted potentiation of the antitubercular effect of a well-known drug Isoniazid in a guinea pig model. M. tuberculosis was not detected in culture from lung and spleen tissues from animals treated by the combined therapy. In addition, we saw minimal histological changes in the lung tissue of animals received Isoniazid in combination with interferon gamma, and they were completely absent in the spleen tissue of animals of this group.
Discussion. During the millennial evolution of the TB pathogen with the host organism, both sides have developed various options for mutual containment, which allows this parasitic system to exist in dynamic equilibrium for a long time. Recent studies have confirmed the results presented in this article, showing that interferon gamma can increase the antibacterial activity of Isoniazid and lead to complete sterilization of cultures, including phenotypically resistant to Isoniazid mycobacteria. Thus, the balance can be shifted towards complete abacillation and a successful clinical outcome of the infectious disease.
Conclusion. Adding the immunomodulatory drug Ingaron in the therapy of drug-sensitive tuberculosis with Isoniazid made it possible to achieve the cessation of bacterial excretion faster; in addition, histological changes in internal organs were less pronounced in animals treated by combination therapy in comparison to animals received only Isoniazid.
Comparison of WGS-based HLA typing bioinformatic tools
Introduction. The widespread distribution of sequencing platforms in research and medical institutions, reduction of sequencing costs and its incorporating to clinical practice make it reasonable to estimate additional parameters on the patient’s sequencing data. Thus, whole-exome and whole-genome sequencing data provide information not only about single-nucleotide polymorphisms, small deletions and insertions, some types of structural variants, but also about the HLA genotype. A mass typing of HLA alleles using advanced bioinformatics tools can be performed based on NGS data. Using the most appropriate tools, the results of HLA typing based on NGS data can contribute to an accurate description of HLA allele frequencies in populations, updating the Allele Frequency Net Database, studying the distribution patterns of HLA alleles within different ethnic groups, and searching for associations with autoimmune diseases.
Aim – a search of the optimal whole-genome-based HLA typing tool for adding it into the bioinformatics data processing pipeline.
Material and methods. Whole genome sequencing with further bioinformatic processing was performed for 150 frozen blood samples. HLA typing of WGS data was performed using tools: xHLA, POLYSOLVER, HLA-LA, HLAscan, OptiType, and Kourami. Libraries for target HLA region sequencing for the same 150 samples were prepared using the NGSgo-MX6-1 primer pool (GenDX, The Netherlands) and the NGSgo-LibrX kit (GenDX, The Netherlands). HLA allele typing on target sequencing data was performed using the NGSengine program.
Results. This study examined the HLA typing accuracy of bioinformatics tools xHLA, OptiType, HLAscan, POLYSOLVER, HLA-LA and Kourami on ≥ 30x whole-genome sequencing data from human samples. HLA typing results were obtained with the NGSgo-MX6-1 kit (GenDX, Netherlands), which were taken as reference results. The POLYSOLVER tool showed the highest accuracy for HLA class I typing; xHLA tool with IMGT/HLA database version 3.22.0 – for HLA class II, POLYSOLVER and OptiType tools require significant time and computing resources, therefore, the bioinformatic tools Kourami and HLAscan are more suitable for wide range HLA typing. All of the bioinformatics tools make more errors for typing HLA class II loci than for typing HLA class I loci, despite that the diversity of HLA class II alleles is significantly lower than of class I. The highest number of incorrectly defined alleles was observed for DQB1 typing.
Conclusion. The results and conclusions obtained in this study provide the basis for a methodological approach to selecting the optimal HLA typing tool for use in bioinformatic pipelines for processing whole genome and/or whole exome sequencing data.
Immunopathogenesis and target therapy of Alzheimer’s disease
Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases in the elderly worldwide. However the ambiguity of the development and causal factors of AD leads to a discrepancy in the understanding of the pathogenesis of the disease and may be one of the reason for the complexity of the development of drugs for AD. The review presents new data on the conduct of clinical trials treatment methods that affect the pathophysiological processes in AD. Targeted drugs aimed at neuroinflammation induced by NLRP3 inflammasome, monoclonal antibodies and drugs for active immunization against beta amyloid, tau protein are considered.