The efficacy of medicinal products based on plasma immunoglobulins and monoclonal antibodies for the treatment and prevention of COVID-19
The development of the COVID-19 pandemic initiated the search for effective drugs for the treatment and prevention of infection. Of particular importance for the fight against the pandemic is the timing of the introduction of drugs into clinical practice. Therefore, countries with developed healthcare systems (EU, USA, Russian Federation, etc.), issue conditional marketing authorizations of drugs for treatment and prevention of COVID-19, based on regulatory requirements for the circulation of medicines in emergency situations. The approval is issued on the basis of limited clinical data, with the condition that the full evaluation of safety and effectiveness will be carried out after the issuance of the authorization in the post-registration period. The COVID-19 pandemic has revolutionized the design and time frame of clinical trials, including phase I, II, and III adaptive trials, which has led to the approval of biologics for the treatment and prevention of COVID-19 in record time in most advanced pharmaceutical countries. At the same time, regulatory/healthcare authorities or international organizations constantly monitor the safety and effectiveness of used drugs and, if necessary, make adjustments (changes to the indications for use, dosage change, drug discontinuation etc.). Since the beginning of the pandemic, in fact, in conditions where the use of medicines was allowed based on very limited data, studies have begun to substantiate the safety and effectiveness of the use of immunoglobulin preparations and monoclonal antibodies for the treatment and prevention of COVID-19. As new data became available, changes were made regarding the indications for use, doses, and other characteristics of preparations of immunoglobulins and monoclonal antibodies for the treatment and prevention of COVID-19. The review presents a critical analysis of the results of evaluation the safety and effectiveness of the use of immunoglobulin preparations and monoclonal antibodies in a pandemic.
Changes in the functional properties of dendritic cells upon contact with microorganisms of the genera Bacillus, Lactiplantibacillus and Limosilactobacillus
Introduction. Previously, we compared the efficacy of phagocytosis and induction of dendritic cell (DC) maturation by 8 different bacteria and yeasts capable of permanent or temporary persistence in the gastrointestinal tract in order to select candidate bacterial vectors for live oral vaccines. It was shown that DCs most effectively absorb microorganisms of the Bacillus, Lactiplantibacillus and Limosilactobacillus genera, and microorganisms of the Bacillus genus more effectively cause DC maturation.
The aim of the study – to evaluate the effect of microorganisms of the genera Bacillus, Lactiplantibacillus and Limosilactobacillus on the functional properties of DCs.
Material and methods. We stimulated human monocyte-derived DCs with inactivated bacteria and assessed cytokine production and the ability of DCs to stimulate of lymphocytes in an allogeneic mixed culture with pure CD4+ T cells.
Results. Microorganisms of the genus Bacillus stimulate the production of interleukin (IL)-6, IL-1β, and IL-10 by DCs. Preincubation of DCs with these bacteria enhances the transformation of CD4+ T-lymphocytes into lymphoblasts and suppresses the production of IL-10 and interferon-γ (IFN-γ) in a mixed culture with T-lymphocytes. DC preincubation with B. licheniformis causes a moderate increase in IL-17 production in a mixed culture, while DC preincubation with B. cereus increases RORc gene expression and also causes a slight increase in the number of CD25+FOXP3+ T cells without a stable increase in FOXP3 gene expression. Lactobacilli weakly stimulate the production of cytokines by DCs and do not affect the ability of DCs to induce blast transformation of CD4+ T cells and generation of CD25+FOXP3+ T cells. DC preincubation with Lactiplantibacillus plantarum causes a slight decrease in IL-10 production in the mixed culture, while DC preincubation with Limosilactobacillus fermentum does not affect the production of this cytokine, but weakens the production of IFN-γ and IL-17.
Conclusion. Microorganisms of the genus Bacillus cause a moderate increase in the stimulating properties of DCs. Lactobacilli have a weak effect on the production of cytokines by DCs and moderately weaken the ability of DCs to stimulate the pro-inflammatory activity of T-lymphocytes.
Expression of Arg1 and MerTK by human macrophages activated by M2-polarizing stimuli and their role in determining low allostimulatory activity
Introduction. One of the universal manifestations of the immunomodulatory activity of M2 macrophages in vitro is the ability to determine a low level of T cell proliferation in a mixed leukocyte culture compared to M1 macrophages. In experimental animals, polarization towards M2 phenotype is largely associated with metabolic reprogramming, in particular, changes in the expression of arginase 1 and tyrosine kinase Mer. However, in humans, the expression and significance of these molecules in determining the low allostimulatory activity of M2 phenotype macrophages remain unexplored.
The aim of the study was to evaluate the expression of Arg1 and MerTK in macrophages polarized by M2-polarizing stimuli in comparison with M1, and to evaluate the role of these molecules in determining the low allostimulatory activity of M2 macrophages.
Material and methods. Macrophages were generated from donor peripheral blood monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) followed by polarization with interferon-γ (IFN-γ) – in M1, interleukin-4 (IL-4) – in M2a, and by interaction in apoptotic cells – in M2(LS) phenotypes. We studied the relative content of MerTK+ and Arg1+ cells, as well as the ability of macrophages to stimulate the proliferation of T-lymphocytes in mixed leukocyte culture (MLC).
Results. Human macrophages generated from circulating monocytes in the presence of GM-CSF express Arg1 and MerTK. The highest content of Arg1+ and MerTK+ cells was found in cultures of macrophages with the M2 phenotype, polarized not only under the influence of IL-4 (M2a), but also as a result of interaction with apoptotic cells (M2(LS)). The expression of Arg1 and MerTK was found to be inversely correlated to the allostimulatory activity of M2 macrophages in MLC. Moreover, blocking these molecules with anti-MerTK antibodies and the arginase 1 inhibitor nor-NOHA leads to an increase in the allostimulatory activity of M2 cells.
Conclusion. The data obtained indicate the expression of Arg1 and MerTK by M2 phenotype macrophages and the involvement of these molecules in determining the low allostimulatory activity of M2 macrophages, which indicates the implication of Arg1 and MerTK in macrophage-mediated modulation of the adaptive T-cell response.
Phenotype and effector functions of GD2-specific CAR-T lymphocytes in vitro
Introduction. The application of T lymphocytes with a chimeric antigen receptor (CAR) underlies new promising technologies for tumor immunotherapy, the main idea of which is associated with the adoptive transfer of modified T cells that are capable of cytotoxicity, this does not require additional costimulation and recognize surface antigens in an MHC-independent manner due to their difference from TCR to receptor structure. This paper presents characterization of CAR-T cells specific for disialoganglioside GD2 ex vivo, which expression is restricted to malignant neoplasms, making this molecule a promising target. The addition of costimulatory domains, such as 4-1BB or CD28 to bone CAR cells increases survival and proliferation of the resulting CAR-T cells, and the insertion of the GITR ligand (GITRL) promotes self-maintenance of the resulting populations and improves antitumor activity.
Aim of the study – to research the phenotype and effector functions of GD2-specific CAR-T lymphocytes in vitro using various functional tests.
Material and methods. Transduced and non-transduced GD2-specific CAR lymphocytes from healthy adult donors were examined for the content of cell memory subpopulations by CD45RA and CD62L markers and cell exhaustion by PD-1 and TIM-3 markers using flow cytometry. Cytotoxicity was studied by the multiplex analysis of conditioned media after cocultivation of the studied genetically modified cells and tumor cell lines on a flow cytometer and by the colorimetric method by the content of lactate dehydrogenase. The effector functions of the resulting CAR-T cells were assessed after cocultivation with target cells for cell activation markers 4-1BB, CD40L, CD69 and FasL using flow cytometry.
Results. Transduced CD4+ and CD8+ lymphocytes represented the least differentiated population of CD45RA+CD62L+ effector cells with high viability and proliferative activity, also, a proportion of exhausted PD-1+TIM-3+ cells was low. The resulting transduced cells exhibited antigen-specific properties for tumor cell lines carrying the target antigen. In addition, the cytotoxic function of transduced T lymphocytes carrying cellular activation markers 4-1BB, CD69 and CD40L, as well as their cytokine-producing activity in the presence of GD2-expressing tumor cells, was observed.
Conclusion. The obtained GD2-specific CAR-T cells exhibit antigen-specific antitumor properties in vitro, which is due to their cytokine-producing activity, increased expression of cell activation markers 4-1BB, CD69 and CD40L, and a low proportion of exhausted PD-1+TIM-3+ cells.
Phenotypic and functional features of in vitro generated TCR-T cells specific for the tumor-associated antigen NY-ESO-1
Introduction. The antitumor activity of T cells holds great potential for the development of immunotherapeutic schemes for the treatment of oncological diseases. Gene modification has become a new milestone in studies of antitumor T-cell immunity, which makes it possible to obtain a population of cells with a T cell receptor (TCR) of a given specificity from a heterogeneous pool of T lymphocytes. In this work, using a gammaretrovirus vector encoding a TCR specific for the epitope of the tumor-associated NY-ESO-1 antigen, NY-ESO-1 specific TCR-T cells were obtained, the features of their phenotype were studied in vitro, and the antigen-dependent activation and antitumor cytotoxic activity.
Aim of the study – to research the phenotypic and functional features of in vitro generated TCR-T lymphocytes for the tumor-associated antigen NY-ESO-1.
Material and methods. The phenotype of transduced cells was studied using flow cytometry by expression of markers of memory cells (CD45RA and CD62L), activation and depletion (CD69, CD95, PD-1, TIM-3). The cytotoxic potential of the obtained cells was assessed using the analysis of the release of lactate dehydrogenase in conditioned media from cultivation with target cells of tumor lines SK-Mel-37 and NW-Mel-38. Additionally, after cultivation with target cells, the expression of markers of cell activation and cytotoxicity (4-1BB, CD69, CD40L, FasL) was assessed using flow cytometry.
Results. Transduced NY-ESO-1-specific TCR-T cells were predominantly poorly differentiated T-lymphocytes with high viability and were characterized by a phenotype shift towards central and effector memory cells compared to non-transduced T-lymphocytes. Shown antigen-specific cytotoxic response to cultivation with tumor cells positive for the expression of NY-ESO-1. When cultured with the SK-Mel-37 line, a significant increase in the proportion of cells expressing CD40L and FasL was observed compared to the proportion in the absence of target cells, which confirms the activation of the effector properties of the transduced T cells in response to the antigen.
Conclusion. In vitro generated NY-ESO-1-specific TCR-T cells are characterized by a poorly differentiated memory phenotype and a high level of viability, and have a cytotoxic potential against tumor lines expressing NY-ESO-1.
Antitumor activity of human macrophages activated by NOD1 and TLR4 receptor agonists in vitro
Introduction. Tumor-associated macrophages play an important role in tumor development by modulating inflammation, immunosuppression and angiogenesis. Depending on the microenvironment, macrophages can exhibit both pro- and antitumor activity. Agonists of pattern recognition receptors, including Toll-like receptors (TLR) and NOD receptors, promote the induction of antitumor activity of macrophages. It is known that combined stimulation of NOD1 and TLR4 receptors in macrophages and other cell types results in a synergistic increase in the expression and production of pro-inflammatory cytokines. However, the effect of a combination of NOD1 and TLR4 agonists on the antitumor properties of macrophages has not been studied.
The aim of the work was to study the mechanisms of antitumor activity of macrophages stimulated by NOD1, TLR4 receptor agonists and their combination in vitro.
Material and methods. The cells of the erythromyeloid HLA-I-negative K562 line labeled with CFSE were used as targets for macrophages. Target cells were cocultured with human macrophages in the presence of a NOD1 agonist, TLR4 agonist, combination thereof or without agonists. K562 cultures without macrophages served as controls. 72 hours after the start of the experiment, the content of viable target cells in cultures was assessed using flow cytometry. To clarify the contribution of soluble mediators and surface molecules to the antitumor activity of macrophages, the effect of activated macrophage supernatants on K562 cells was studied, and macrophages and K562 cells were separated using semipermeable membranes. Neutralizing antibodies were used to assess the contribution of TNF and IFN-β to the antitumor effect of macrophages. NADPH oxidase was inhibited with apocynin.
Results. Macrophages not activated by NOD1 or TLR4 agonists enhanced the growth of K562 tumor cells compared to the control. Separate NOD1 and TLR4 agonists abolished the protumor effect of macrophages, while stimulation of macrophages with a combination of agonists led to a multiple decrease in the number of K562 cells compared to the control. In the absence of macrophages NOD1 and TLR4 agonists did not affect the number of K562 cells. The antitumor effect of macrophages activated by a combination of NOD1 and TLR4 agonists was partially abolished by anti-TNF antibodies and when macrophages and K562 cells were separated by a semipermeable membrane. It has been shown that one of the components of the antitumor activity of macrophages activated by a combination of NOD1 and TLR4 agonists is an antiproliferative effect.
Discussion contains an analysis of the possible mechanisms of the antitumor effect of macrophages activated by a combination of NOD1 and TLR4 agonists.
Conclusion. The possibility of switching the activity of human macrophages from protumor to antitumor under the action of a combination of NOD1 and TLR4 agonists in vitro has been shown. To implement the antitumor response of macrophages against K562 cells, TNF secretion and direct intercellular interaction between K562 and macrophages are required. The results obtained allow to consider the combination of NOD1 and TLR4 agonists as a potential anticancer drug.
Differences in immune response of C57BL/6, В10.D2 (R101) and BALB/c mice to EL-4
Introduction. The immune system provides antitumor immunity, or immune surveillance. Syngeneic adoptive immunotherapy is often used for cancer therapy, but it is ineffective due to the weak immunogenicity of tumor antigens for the own immune system. In theory, allogeneic adoptive immunotherapy should be significantly more effective.
Aim – to study the immune response to EL-4 in C57BL/6, B10.D2 (R101) and BALB/c mice and evaluate the possibility of syngeneic and allogeneic adoptive immunotherapy.
Material and methods. In the study, we used C57BL/6, BALB/c and B10.D2 (R101) mice. Allogeneic mice BALB/c and B10.D2 (R101) were immunized with EL-4 cells intraperitoneally, syngeneic C57BL/6 mice – intradermally with tight ligation of the tumor in the future and reimmunization in 2 months. To assess the immune response to a tumor in the spleen, the cellularity and subpopulations of T-lymphocytes and NK cells were determined on days 3, 12, 15 by flow cytometry.
Results. The primary immune response to EL-4 in syngeneic C57BL/6 mice consisted of an increase in the amount of T-cell subpopulations (central memory T helpers and central memory CTLs), and NK cells in the spleen on day 7. During the secondary immune response, there was a decrease in the amount of T-lymphocytes, T-helpers, and effector memory cells (CD4+-TEM and CD8+-TEM) in the spleen in the early periods after reimmunization, with a further increase in all CTLs, including subpopulations of central and effector memory CTLs after a week. Moreover, the amount of effector memory CTLs in the spleen by day 7 exceeded 2.5 times compared with the control. The amount of natural killers increased by the end of the 1st week and was raised up to 12 days. None of the reimmunized mice showed tumor growth during the observation period (400 days). After administration of EL-4 to allogeneic B10.D2 (R101) mice we detected a decrease in almost all the studied cell subpopulations in the early periods in the spleen. An increase in the amount of CTL was detected on the 12th day, and the level of NK cells was raised by the 13th day. In allogeneic BALB/c mice, a decrease in the amount of all studied cell subpopulations was also observed in the early periods after immunization. We noted an increase in the number of T-lymphocytes from the end of the 1st week and persisting up to 13 days, and an increase in the amount of T-helpers and CTL from 7 to 11 days in BALB/c mice.
Conclusion. We showed that the maximum expansion of effector cytotoxic T-lymphocytes in syngeneic C57BL/6 mice was on day 7 and it was later in allogeneic mice: on day 11 in BALB/c mice and on day 12 in B10.D2 (R101) mice. Therefore, the greatest therapeutic effect can be expected from the transfer of CTL effector subpopulations for adoptive immunotherapy during these periods.
Expression of the TNFSF13B, APRIL, VEGF, FGF1 и EGF genes in different phenotypes of chronic rhinosinusitis with nasal polyps
Background. Chronic Rhinosinusitis with Nasal Polyps (CRSwNP) is a chronic hyperplastic inflammatory process based on remodeling in the mucosa membrane of the paranasal sinuses leading to the formation of polyps. According to modern concepts, various growth factors can take part in the remodeling of the respiratory tract.
The aim – to study the level of growth factors genes expression in nasal polyps’ tissue in different phenotypes of CRSwNP.
Material and methods. 72 patients with bilateral CRSwNP were divided into 3 groups according to disease phenotype each of 24 patients: Group I – CRSwNP without asthma (BA) and allergy; Group II – CRSwNP with allergic BA; Group III – CRSwNP with nonallergic BA. Comparison group – patients with hypertrophic rhinitis without BA and atopy. We used real-time polymerase chain reaction in polyp’ tissue to study the levels of genes expression encoding proteins EGF, VEGF, FGF, BAFF and APRIL.
Results. The expression of genes TNFSF13B и FGF1 (encoding factors BAFF and FGF) in all CRSwNP phenotypes was statistically significantly higher than in the comparison group. Among the phenotypes of CRSwNP these growth factors were significantly lower in patient with CRSwNP and allergic BA. The expression of VEGF and APRIL in nasal polyps in all groups was lower than in the comparison group without statistically significantly. The expression of the gene encoding EGF was detected only in the comparison group, whereas the expression of this gene was not detected in the polyps’ tissue.
Conclusion. We demonstrated an increase of TNFSF13B и FGF1 genes in polypous tissue in all CRSwNP phenotypes, while the expression of EGF, VEGF and APRIL genes in comparison group was significantly lower or wasn’t detected. These data indicate the involvement of BAFF and FGF in the formation and growth of polyps. BAFF in the polypous tissue promotes the maturation of B-lymphocytes and their production of immunoglobulins. As the same time, FGF induces tissue fibrosis. High expression level of TNFSF13B and FGF1 genes suggest that their protein products – BAFF and FGF – can be considered as predictive biomarkers of the formation and recurrence of nasal mucosal polyps and paranasal sinus polyps, especially in the absence of atopy.
It is worth noting separately that all the patients with CRSwNP have the lowest expression of EGF gene detected in the polyp tissue. Therefore, the participation of EGF in polyp formation seems unlikely. Perhaps there isn’t the epithelial damage in the processes. VEGF and APRIL are responsible for hypertrophy of the nasal mucosa and paranasal sinuses, but do not cause polyp formation, regardless of the presence of atopy. This indicates that the molecular mechanisms of CRSwNP associated with allergic BA have their own characteristics that require additional research.
Сorrelation between age and the intensity of the post-vaccination humoral immune response in individuals passed COVID-19
Introduction. Despite widespread vaccination against COVID-19 worldwide, the epidemiological situation remains insufficient. The appearance of mutant forms of SARS-CoV-2 with increased virulence causes new waves of COVID-19 morbidity. The virus variability can cause the reduction of the vaccination effectiveness. In this regard, it is important to study the dynamics of post-vaccination immunity, its features in various population groups, as well as the relationship between post-infectious and post-vaccination immunity. The study continues a series of researches of the features of SARS-CoV-2-specific immunity in COVID-19 convalescents and recipients of the «Sputnik V» vaccine.
The aim of the study – assessment of the intensity of the SARS-CoV-2-specific immune response in different age groups of people passed COVID-19 and subsequently vaccinated with «Sputnik V».
Material and methods. With the help of EIA, the level of IgG antibodies to S-antigen and N-antigen of SARS-CoV-2 was studied in 155 samples of blood sera of individuals (men and women) aged 18 to 70 years who passed COVID-19 and after 6 month of recovery underwent a full course of vaccination with «Sputnik V». Samples from individuals of the following age groups were examined: from 18 to 35, from 35 to 45, from 45 to 55, from 55 to 65, from 65 to 70 years.
Results. In all the examined age groups of individuals passed COVID-19 and then vaccinated with «Sputnik V», there was a high level of IgG antibodies to SARS-CoV-2 S-protein (positivity coefficient 8.1 and higher). The level of IgG antibodies to the N-protein in more than half of the cases was lower than positive values among all the subjects.
Conclusion. The study showed that individuals passed COVID-19 and then vaccinated with «Sputnik V», regardless of age, have intense post-vaccination humoral immunity.
Lipid metabolism in experimental autoimmune atherosclerosis
Introduction. The current paradigm regarding the pathogenesis of atherosclerosis is that there is a causal relationship between dyslipidemia and the formation of atherosclerotic plaque. In studies of the last few decades, convincing data have been obtained regarding the involvement of autoimmune reactions to lipoproteins in the pathogenesis of atherosclerosis. However, the relationship between the autoimmune reaction to lipoproteins, lipid metabolism, and atherosclerotic lesions remains unclear.
The aim of the study was to examine lipid metabolism in rats and rabbits with atherosclerosis-like aortic changes induced by immunization with native human high-density lipoproteins (nhHDL).
Material and methods. We compared the lipid profile of blood plasma and peripheral blood mononuclear cells (PBMC) in nhHDL/IFA-immunized, incomplete Freund’s adjuvant (IFA) injected, and intact rats and rabbits, as well as the production of inflammatory and proatherogenic cytokines TNFα and PDGF-BB by periaortic adipocytes in rabbits.
Results. Changes in the lipid profile of blood plasma and PBMC in animals immunized with nhHDL/IFA and in IFA-injected animals were similar, while atherosclerosis-like changes in the aortic wall were observed only in the nhHDL/IFA-immunized animals. Consequently, the changes in lipid metabolism of rabbits and rats immunized with nhHDL/IFA were induced by the IFA and these changes were not associated with atherosclerotic aortic lesions. Production of TNFα and PDGF-BB by periaortic adipocytes in rabbits that were immunized with nhHDL/IFA and developed atherosclerosis was the same as that of intact rabbits.
Conclusion. We found no evidence that changes in lipid metabolism are necessary for the development of atherosclerotic aortic lesions induced by the immune response to HDL.
Clinical and immunological characteristics of the postcovid period in patients with moderate-severe COVID-19 who received therapy with the inclusion of an IL-6 receptor antagonist
Introduction. Significant progress in clinical research has led to a better understanding of the pathogenesis of COVID-19, contributing to its more effective therapy. The revealed key role of IL-6 in the development and intensification of the «cytokine storm», which has received reliable pathophysiological and pharmacological justification, supports the use of therapeutic strategies aimed at IL-6 or its receptor. Nevertheless, the changes that occur in the immune system after COVID-19 are of interest for a better understanding of the mechanisms of the formation of postcovid syndrome in patients receiving therapy with the inclusion of IL-6 receptor antagonists, its pathogenesis and the search for targets for further therapeutic effects.
The aim of the study – to study the clinical and immunological parameters of patients who underwent a moderate-severe variant of COVID-19 and received therapy with the inclusion of IL-6 receptor antagonist during treatment and 6 months after discharge/recovery.
Material and methods. 30 hospitalized patients were examined with the diagnosis: COVID-19, moderate-severe form; complication: bilateral polysegmental pneumonia. The comparison group consisted of practically healthy donors (30 people). The dynamics of laboratory parameters (general clinical, biochemical and immunological) were evaluated against the background of therapy with IL-6 blockers at discharge, as well as 6 months after discharge from the hospital, in addition, the quality of life was assessed 6 months after the COVID-19.
Results. Postcovid syndrome in patients who have undergone COVID-19 in a moderate-severe form manifests itself in a number of symptoms – decreased appetite, weakness, sleep disorders, depression, headache, shortness of breath, cough, tachycardia, dizziness. In addition, there is an exacerbation of chronic diseases that requires correction in planned therapy. 6 months after discharge from the hospital, there is an increase in CRP and fibrinogen levels, which most likely also reflects the deterioration of the course of concomitant pathology. There is an increase in the level of T-lymphocytes, as well as a decrease in the level of B-lymphocytes.
Conclusion. Patients who had COVID-19 require dynamic follow-up. In patients with a moderate-severe form of COVID-19 who received complex therapy with the inclusion of monoclonal antibodies to IL-6 receptors, dexamethasone, antiviral therapy, anticoagulants, oxygen therapy, there are dysregulatory processes in the immune system that persist 6 months after recovery.