Introduction. Adenovirus (Ad) vaccines are successfully used for immunization against some tumor-associated antigens, as well as a number of infectious pathogens. Dendritic cells (DC) are professional antigen-presenting cells, so transduction of these cells by Ad vectors is especially important for the effective action of Ad vaccines.

The aim of the study was to determine the efficiency of transduction of human DCs by adenovirus vectors of different serotypes.

Material and methods. Peripheral blood mononuclear cells of healthy donors (n = 11) were isolated by centrifugation in a Ficoll-Verografin density gradient. Monocytes were enriched from the mononuclear fraction of blood by negative immunomagnetic separation using the Dynabeads kit. For differentiation into DCs, monocytes were cultured in vitro for 7 days in the presence of a cytokine mixture containing 70 U/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 50 U/ml IL-4. As a result, the differentiated cells expressed CD11c and HLA-DR markers at a high density, indicating DC maturation. The resulting cells were infected with recombinant Ad vectors carrying a green fluorescent protein (GFP) reporter gene. Three serotypes were tested: Ad5, sAd23, and Ad26, as well as a chimeric Ad5 vector containing the corresponding domain of the Ad35 serotype at the C-terminus of its fiber (Ad5/35). The percent of transduced B lymphocytes and the mean fluorescence intensity (MFI) of the GFP reporter protein were determined 48 h after infection. After transduction, expression of CD86 and HLA-DR antigens was determined on GFP⁺ and GFP^– cells.

Results. High Ad doses (from 60 to 1000 PFU/cell) provided transduction of more than 90 % of DCs. In this case, the fluorescence histograms of GFP⁺ cells had a unimodal character, which was clearly separated from the fluorescence of GFP^- cells. With a decrease in the Ad dose, the character of the histograms obtained during infection with different serotypes differed from each other. During infection with Ad5, Ad26, and Ad5/35 serotypes, with a decrease in the virus dose, a decrease in transduction was manifested mainly in a drop in the intensity of GFP fluorescence. Further reduction of the Ad dose, when only a part of DCs was transduced, led to a significant drop in the proportion of GFP⁺ cells, with a moderate decrease in MFI. When DCs were infected with sAd23, the decrease in the fluorescence level and the percent of GFP⁺ cells occurred simultaneously with a decrease in the virus dose. Dose-dependent transduction curves were obtained for 11 volunteers. Comparison of the transduction efficiency of different serotypes showed that the Ad5/35 and Ad26 vectors had the highest transducing capacity. To transduce 50 % of DCs, Ad5/35 and Ad26 doses of about 1 PFU/cell were required. The Ad5 and sAd23 vectors showed the lowest efficiency. To transduce 50 % of DCs, Ad5 and sAd23 doses of about 140 and 70 PFU/cell were required, respectively. Adenoviral transduction increased the expression of CD86 and HLA-DR antigens on DCs.

Conclusion. Ad vectors have a high transducing capacity for DCs. Transduction of DCs with Ad5, Ad26, and Ad5/35 serotypes provides high transgene expression. Adenoviral transduction induces the transition of DCs to a more mature form.

Introduction. Recently, an alternative variant of IL-5 (IL-5d2) has been identified. Full-length IL-5 plays an important role in the maturation, activation and migration of proinflammatory eosinophil cells, while the biological role of IL-5d2 still unknown.

Therefore, the aim of this study was to investigate the biological effects of a new IL-5d2 isoform in vitro.

Material and methods. Full-length IL-5 and its novel isoform IL-5d2 were produced in HEK293T cells by transfection with plasmids carrying the corresponding genes. The concentration of IL-5 and IL-5d2 was determined by enzyme-linked immunosorbent assay (ELISA) using commercial monoclonal and polyclonal antibodies. Eosinophils were obtained by differentiation of BALB/c mouse bone marrow cells in the presence of growth factors GM-CSF, IL-3, and IL-5 or IL-5d2. Cytopreparations were prepared from the cells, followed by differential staining with azure and eosin and quantitative calculation of the proportion of eosinophils by light microscopy.

Results. The differentiation of eosinophils occurred best in the presence of all 3 factors (GM-CSF, IL-3 and IL-5). Replacing the full-size IL-5 with its splice variant led to a slowdown in the differentiation of eosinophils from mouse bone marrow cells. It should be noted that over the same period of time (5 days), the number of eosinophils in the presence of the splice variant IL-5d2 of the mouse decreased by 25 % compared with the full-size form of IL-5 of the mouse. Thus, the biological activity of the splice variant IL-5d2 of the mouse is lower compared to the full-size IL-5 of the mouse. Additional experiments have demonstrated that differentiation of eosinophils can occur only during incubation with IL-5 in the absence of GM-CSF and IL-3. The shortened isoform of IL-5d2 also contributed to the differentiation of eosinophils. Further, the ability of IL-5d2 to competitively inhibit IL-5-dependent differentiation of eosinophils was studied. As a result, it was shown that IL-5d2 doesn’t act as an inhibitor of the activity of full-sized mouse IL-5.

Conclusion. Thus, new isoform IL-5d2 exhibited own in vitro biological activity similar to the full-length IL-5. In addition, IL-5d2 does not inhibit IL-5-dependent eosinophil differentiation.

Introduction. Adenovirus vectors can induce a humoral and cellular immune response to target antigens. However, enhancing their immunogenicity is sometimes essential, requiring new approaches to produce adenovirus-based vaccines.

Aim – to study the ability of genetic constructs that act as adjuvants to increase the immunogenicity of the protective antigen (PA) of Bacillus anthracis expressed in a recombinant adenovirus vector.

Material and methods. Adenoviruses carrying adjuvant constructs with the model antigen (Ad5-Ii-PA and Ad5-Ii-fur-PA) were obtained by molecular cloning and transfection of the eukaryotic cell line. Antigen expression was confirmed by Western blotting. The resulting adenoviruses were used to immunize female BALB/c mice. The mice blood serum was collected on the 28^th day after adenovirus immunization. The specific antibodies to the antigen were detected by ELISA.

Results. A single intramuscular immunization of laboratory animals with a 1 - 10⁷ PFU dose of Ad5-Ii-PA and Ad5-Ii-fur-PA demonstrated an increase in immunogenicity compared to the control adenovirus carrying only the secretable form of PA’s fourth domain. Ad5-Ii-fur-PA [geometric mean titer (GMT): 2539.84; confidence interval (CI) 95 %: 1744–3698] produced the highest antibody titer compared to Ad5-Ii-PA (GMT: 1425.44; CI 95 %: 824–2465) and Ad5-sPA (GMT: 634.96; 95 % CI 436–924). This is presumably due to the ability of the Ii-PA complex to be secreted onto the cell surface, providing access for antigen-presenting cells.

Conclusions. Adenoviral vectors Ad5-Ii-PA and Ad5-Ii-fur-PA, carrying fusion constructs containing the invariant chain and the invariant chain with a furin site as adjuvants, were constructed. The obtained recombinant adenoviruses demonstrated a reliable increase in immunogenicity compared to the adenovirus carrying only secretable PA. Ad5-Ii-fur-PA exhibits the highest immunogenicity.

Introduction. In recent years, adenovirus vaccines have firmly established themselves as one of the leading platforms for creating new vaccines. The advantages of adenovirus vaccines have recently become most evident during the COVID-19 pandemic, which was caused by the global spread of the SARS-CoV-2 coronavirus. However, adenovirus vaccines have one feature that does not allow us to talk about these vaccines as an absolutely neutral carrier. This feature is associated with the existence and possible influence of anti-vector antibodies on the development of the immune response. There is conflicting data on the possible influence of anti-vector antibodies on the success of vaccination with adenovirus vaccines.

The aim of the study was to compare two methods for detecting vector-neutralizing antibodies.

Material and methods. The study included 20 volunteers (8 men and 12 women aged 18 to 70 years, median 23.5 years). All volunteers were vaccinated with two doses of the «Sputnik V» vaccine between January and February 2021. The first dose was rAd26-based, followed by the second dose 21 days later, based on rAd5. Approximately 9 months after the primary vaccination, revaccination was performed using the rAd26-based «Sputnik Light» vaccine. Serum samples were collected immediately before (time point T1) and one month after booster vaccination (time point T2).

Two variants of the rAd26 vector were used in the study. One variant contained the reporter gene GFP encoding the green fluorescent protein GFP (rAd26-GFP), and the other variant contained the gene of the full-length S-protein of the wild-type SARS-CoV-2 (rAd26-Spike). In the vector neutralization test, human lung carcinoma A549 cells were used as target cells. Transduction of cells by rAd26-GFP was determined by counting the proportion of GFP⁺ cells and by determining the proportion of Spike⁺ cells by rAd26-Spike using the monoclonal antibody XR15 against the S-protein.

Results. Vector-neutralizing activity was first measured using the rAd26-GFP vector. Sera from vaccinated volunteers dose-dependently inhibited the entry of the rAd26-GFP vector into A549 cells. Sera collected before booster vaccination had significant vector-neutralizing activity (median = 156), which was higher than the baseline values. After booster vaccination, the level of vector-neutralizing antibodies significantly increased relative to the values before revaccination (median = 606; T1 vs T2 p < 0.001). Using the rAd26-Spike vector results were obtained that were largely consistent with the data obtained using rAd26-GFP. A high correlation was found between the results obtained using the rAd26-GFP and rAd26-Spike vectors, which was further increased by blocking anti-Spike antibodies using a recombinant Spike protein preparation.

Conclusion. The method using the rAd26-GFP vector is simpler, and its results do not depend on the presence of anti-S-antibodies in the serum sample. The method using the rAd26-Spike vector seems to be more labor-intensive, requiring additional blocking of anti-S-antibodies. However, this method allows the use of the vector directly from the vaccine preparation, and the results obtained by this method are more consistent with the real situation.

Introduction. One of the key manifestations of immunosenescence is the inflammaging, which can lead to both successful aging and longevity, and to pathological aging and the development of age-associated pathology. Perhaps the path followed by inflammatory aging will depend on the ratio of systemic proinflammatory/anti-inflammatory mediators in the elderly.

Aim – assessment of the levels of pro- and anti-inflammatory cytokines in centenarians and senile age persons with various aging phenotypes.

Material and methods. 80 senile age persons, 100 centenarians and a comparison group consisting of 50 young people were examined. Within the senile age and centenarian age groups, physiological and pathological aging phenotype subgroups were divided based on the Charlson Comorbidity Index, the Short Physical Performance Battery and the Mini-Mental State Examination. The levels of cytokines IL-1β, IL-4, IL-6, IL-10, IL-18, TNFα, TGFβ in the blood plasma of the study groups was determined by enzyme immunoassay using «Vector-Best» (Russia) and CloudClone Corp. (USA) kits. The obtained data were evaluated using the Statistica 14 software package (StatSoft, EU) and GraphPad Prizm (Prizm, USA).

Results. In centenarians, an increase in levels of pro-inflammatory cytokines IL-6, TNFα, IL-1β, IL-18 and anti-inflammatory cytokines IL-10, TGFβ was shown. In senile age persons an imbalance of cytokines was revealed, manifested in an increase in the level of IL-6 and a decrease in levels of the anti-inflammatory cytokines IL-10 and TGFβ. An increase in the level of IL-4 can be considered as a marker of the phenotype of successful aging, and an increase in the levels of IL-6 and TNFα – as markers of the phenotype of pathological aging in senile age persons and centenarians.

Conclusion. The identified markers of pathological and successful aging phenotypes will help to predict the development of age-associated pathology or longevity.

Introduction. Allergy diagnostics are based on patient history, skin and provocation tests, and the determination of specific IgE levels in serum. However, there are cases where patients exhibit symptoms of an allergic disease without detectable specific IgE to allergens, such as in local allergic rhinitis (LAR). Studying allergen-specific B-lymphocytes in such patients may help to accurately diagnose hypersensitivity.

The aim of the study is to develop and optimize a method for detecting allergen-specific IgE⁺-B-cells in the blood of allergic patients.

Material and methods. The study included 6 adult patients with birch pollen allergy, 1 patient with LAR and 5 healthy adult volunteers without allergic diseases. Enzyme-linked immunosorbent assay, basophil activation test (BAT), flow cytometry, and special protocols for staining and analyzing B-lymphocytes were used to develop and optimize the detection method.

Results. It was found that patients with birch pollen allergy have detectable allergen-specific IgE⁺-B-lymphocytes during the pollen season. The patient with LAR also had identifiable specific B-lymphocytes, suggesting the potential involvement of these cells in the development of allergic reactions even in the absence of specific IgE in serum and basophil activation by Bet v 1 allergen in blood samples.

Conclusion. The developed methods and approaches allow for more accurate diagnosis of allergic reactions, including cases where routine allergological examination methods do not detect specific IgE antibodies. Identifying allergen-specific IgE⁺-B-cells in patients’ blood opens new possibilities for studying the pathogenetic mechanisms of development and diagnosis of allergic diseases.

Introduction. The studies conducted in the recent decades have confirmed an important role of vitamin D in the immune response to TB infection. However, contradictory results of cholecalciferol applications obtained by clinical studies kept us from a clear conclusion about effectiveness of vitamin D in TB treatment.

Aim – to evaluate cholecalciferol influence on the synthesis of β1- and β2-defensins in the dynamic observation of adolescents with advanced TB.

Material and methods. A cohort prospective study included 33 patients (aged 13–17 years) admitted to the Adolescents’ Department of the Central TB Research Institute due to advanced pulmonary TB. The concentration of vitamin D in blood serum was determined by chemiluminescence immunoassay on the Maglumi 2000 Plus automated analyzer (Snibe, China). The levels of β1-defensin were determined by enzyme immunoassay using the SEB373Hu reagent kit (Cloud-Clone Corp., China), β2-defensin – SEА072Hu (Cloud-Clone Corp., China). In cases of expressed calcidiol deficiency (< 10 ng/mL) the dotation of vitamin D was 7000 IU daily; in cases of calcidiol deficiency (10 to 20 ng/mL) – 5000 IU daily. After one month of vitamin D administration the levels of 25(ОН)D, β1- and β2-defensins were determined again, a prophylactic dose of cholecalciferol was administered (1000 IU daily for 5 months) followed by control studies. The dynamic observation was conducted during 6-month chemotherapy: sputum microbiological studies, clinical and X-ray examinations. The comparison of vitamin D, β1- and β2-defensins levels in the dynamics was conducted using paired sample t-test. The differences were considered significant at p ≤ 0.05.

Results. Out of 33 patients, vitamin D deficiency was established in 42.4 % (14/33), expressed deficiency – 57.6 % (19/33) of patients. AMPs (n = 33): β1-defensin was 6.67 ± 0.83 ng/mL, β2-defensin – 0,833 ± 0,15 ng/mL. After one month of therapeutic dotation of cholecalciferol we observed statistically significant growth of levels of calcidiol – 9.35 ± 0.61 and 42.24 ± 2.14 ng/mL (р = 0.0001); β1-defensins – 6.67 ± 0.83 and 8.27 ± 1.15 ng/mL (р = 0.026); β2-defensins – 0.833 ± 0.15 and 1.01 ± 0.17 ng/mL (р = 0.037). On achieving the adequate levels of vitamin D within one month, subsequent administration of prophylactic cholecalciferol did not show any statistically significant differences in the levels of 25(ОН)D, β1- and β2-defensins after 2 and 6 months of observation. We observed sputum conversion in all 8 sputum positive patients within 3 months of TB treatment. Positive X-ray dynamics was observed in all 33 patients within 2–6 months of observation: resolution and/or consolidation of infiltration or foci, cavity closure or reduction.

Conclusion. It is expedient to administer vitamin D dotation to patients with advanced pulmonary TB to achieve its adequate level and induction of β1- and β2-defensins synthesis. Further clinical trials are necessary to study effectiveness of vitamin D administration and determination of optimal doses for treatment and prevention of TB.

Introduction. One of the directions of adoptive cancer immunotherapy is the use of T-lymphocytes expressing the chimeric antigen receptor (CAR) for the treatment of hemoblastosis and solid tumors. Genetic modification of lymphocytes can be achieved by transduction of retroviral genes, which leads to constitutive expression of CAR in T cells, or by electroporation of plasmid/mRNA, which leads to temporary expression of immunoreceptors, minimizing the risk of potential autoaggression and genome disturbance. We have developed the drug CAR-CEA, which is a lymphocyte modified by electroporation of plasmid DNA encoding CAR, directed against carcinoembryonic antigen (CEA).

Aim – to study the pharmacokinetics of CAR–CEA with a single intravenous administration to experimental animals and to evaluate the half-life of the drug.

Material and methods. The study was performed on a B6D2F1 mouse model. CAR-CEA was obtained by transfecting mouse lymphocytes with plasmid 3C1-3g electroporation, and administered intravenously at a dose of 10⁶ cells per animal. The amount of CAR-CEA in blood cells, lung, spleen and thymus tissues was assessed by PCR based on the content of plasmid DNA. The parameters of the pharmacokinetics of CAR-T were evaluated at 14 time points (30 min – 30 days).

Results. The maximum concentration of CAR-CEA plasmid DNA in the whole blood of mice was determined 30 minutes after administration and was 125 ± 29 pg/ml. Further, the decrease in concentration occurred exponentially. The half-life was 105 ± 7 hours. CAR-CEA showed the ability to circulate in the bloodstream for up to a month. The most significant and prolonged accumulation of the drug was observed in the spleen.

Conclusion. The results of our preclinical study show that CAR-CEA can be used for anti-СEA therapy under the condition of repeated injections.

Introduction. The formation of specific antibodies to benzo[a]pyrene (BP) reflects the state of the protective reactions of the immune system and metabolic and repair processes in humans. It may be promising to create a personalized method of diagnosing cancer risks based on the immune mechanisms of human adaptation to chemical carcinogens.

Aim – to study the associations between the formation of antibodies to BP, harmful environmental factors, mutations in tumor suppressor genes and the risk of breast cancer (BC) in young age women.

Material and methods. We studied 155 young age women (25–40 years old) with a primary diagnosis of invasive breast carcinoma of a nonspecific type and 111 women without BC. Data on systemic obesity, smoking, stove heating, urban environment, and harmful factors in the work environment were obtained from medical records and personal dates. Analysis of idiotypic IgA and IgG antibodies specific to BP was determined in the blood serum of women using a non-competitive semi-quantitative enzyme immunoassay. Typing of mutations BRCA1 5382insC (rs80357906), 4153delA (rs80357711) and BRCA2 6174delT (rs80359550) genes were performed by real-time PCR followed by melting analysis.

Results. BC patients and women in the comparison group were differed in the median level of IgG-BP in the blood serum (p < 0.001), smoking (p = 0.002) and the frequency of occurrence of the 5382insC BRCA1 mutation (p = 0.016). BC patients more often had high levels of IgG-BP (> 3.5 CU) in the blood serum (77.7 vs 52.3 % in the comparison group, χ² = 14.28; d(f) = 1; p = 0.0002), women in the comparison group – low levels of IgG-BP (< 3.5 CU). The cooperative effect of high serum IgG-BP levels, environmental factors (smoking, urban environment, and obesity) and mutations BRCA1-2 on the risk of BC were studied using a stepwise logit regression model. We identified significant synergistic associations of high serum levels of IgG-BP (OR = 3.66; 95 % CI 1.90–7.08, p = 0.0001) and systemic obesity (OR = 5.13; 95 % CI 1.57–16.75, p = 0.006) with the risk of BC. Factors such as individual immune responses to BP, the mutations BRCA1-2, smoking and urban environment were not associated with each other in young age women with BC.

Conclusion. Patients with BC more often had high levels of BP specific IgG antibodies, smoked and were carriers of the 5382insC BRCA1 mutation compared to healthy women. The formation of antibodies to BP in young age BC-patients was associated with obesity. Screening of mutations 5382insC and 4153delA of the BRCA1 gene in young age BC-patients can make adjustments to further tactics of their treatment.

Introduction. Monocytes refer to cells of innate immunity. In 2010 the International Union of Immunological Societies Nomenclature Committee approved the division of monocytes into 3 subpopulations based on the expression of high-affinity receptor to lipopolysaccharide (CD14) and low-affinity receptor to IgG (CD16): classical (MO1), intermediate (MO2) and non-classical monocytes (MO3). Given the important role of monocytes as components of the microenvironment of chronic lymphocytic leukemia (CLL) cells, as well as the currently available contradictory data in the assessment of monocyte subpopulations, further study of the subpopulation composition of monocytes depending on the stage of CLL and the current therapy deserves attention.

The aim of the study is to analyse the subpopulation structure of monocytes in the peripheral blood of patients with CLL.

Material and methods. The material for the study was whole venous blood stabilized with anticoagulant K₂-EDTA. The study included 30 donors and 196 patients diagnosed with CLL (50 with primary diagnosed, untreated CLL and 146 patients on therapy). To assess the subpopulation composition of blood monocytes we used a panel of Mab conjugates including CD3-FITC, CD56-PE, CD16-PE-Cy7, CD14-APC and CD45-APC-Cy7 (BD Biosciences, USA, and Beckman Coulter, USA). We determined 3 subpopulations of monocytes: MO1 (CD14⁺⁺CD16^–), MO2 (CD14⁺⁺CD16⁺) and MO3 (CD14⁺CD16⁺⁺).

Results. The study of blood monocyte subpopulation showed that the main subpopulation of peripheral blood monocytes in CLL is represented by MO1. Patients with CLL on therapy not containing ibrutinib showed a significant decrease in the relative content of the MO1 compared to other examined groups of patients. Also, there was a significant increase in the relative content of the MO2 subpopulation in all groups of CLL patients in relation to the donor group. Analysis of the relative content of the MO3 subpopulation revealed a significant increase in its value in patients on ibrutinib-free therapy in comparison with donors and groups of patients with CLL treated with other regimens.

Conclusion. In all groups of patients with CLL, changes in the subpopulation of monocytes were observed due to an increase in the relative content of intermediate forms (MO2). Decrease in the relative content of MO1 and increase in the relative content of MO3 were observed in patients receiving therapy without ibrutinib. Analysing the absolute content of monocyte subpopulations in CLL in the studied groups did not reveal significant differences.

Introduction. Determination of anti-D-antibodies content is practiced both in perinatal medicine during the pregnancy management for women with negative Rhesus-factor, and in pharmaceutical industry to assess of specific activity of human anti-D-immunoglobulin and donor plasma for its production. Existing differences in data interpretation indicate the relevance of their unification. A standardized analytical method is necessary. The indirect hemagglutination reaction in gel is the most accessible in implementation.

The aim was to develop a method for quantitative determination of anti-D-antibodies by indirect hemagglutination reaction in gel.

Material and methods. Human immunoglobulin preparations and donor blood plasma were tested. The reaction was carried out in ID Coombs Anti-IgG gel cards. Quantitative assessment was carried out in relation to the standard sample.

Results. The procedure for application of a standard sample and calculating of the concentration of anti-D-antibodies are proposed. The criteria for acceptance of the results are defined. The reliability and reproducibility of the results has been confirmed.

Conclusion. A standardized method for quantitative determination of anti-D-antibodies by indirect hemagglutination reaction in gel, which allows expressing the result in international units, has been developed.