With the onset of the COVID-19 pandemic, a number of molecular-based tests have been developed to diagnose SARS-CoV-2 infection. However, numerous available serological tests lack sufficient sensitivity or specificity. They do not detect specific antibodies in a significant proportion of patients with PCR-confirmed COVID-19. There is evidence that some convalescents have a relatively short-lived humoral immunity. In contrast, a number of publications have shown that T-cell response to human coronaviruses, including SARS-CoV-1, MERS, and SARS-CoV-2, can be strong and long-term. Assessment of T-cell immunity to SARS-CoV-2 is important not only for stratification of risks and identification of potentially protected populations with immunity acquired as a result of previous infection, but also for determining immunogenicity and potential efficacy of vaccines under development. The existing methods of quantitative or semi-quantitative assessment of specific T-cell response are mainly used in scientific research and are not standardised. The aim of the study was to develop and verify experimentally a test kit to be used in a standardised procedure for in vitro determination of T-cells specific to SARS-CoV-2 antigens, in human peripheral blood. Materials and methods: the TigraTest^® SARS-CoV-2 kit developed by GENERIUM, which determines the number of T-cells secreting interferon gamma in vitro, was tested in the study. Samples of venous blood of volunteers from three different groups were analysed in the study: presumably healthy volunteers; COVID-19 convalescents; individuals vaccinated against SARS-CoV-2. Results: the authors developed the TigraTest^® SARS-CoV-2 kit for in vitro determination of T-cells specific to SARS-CoV-2 antigens in human peripheral blood, demonstrated its specificity and performed preliminary assessment of its sensitivity. The study analysed the range and magnitude of the T-cell response in convalescent and vaccinated individuals. A pronounced T-cell response was also shown in some individuals with no symptoms or with unconfirmed diagnosis. It was discovered that the mean T-cell response to peptides of the spike protein (S-protein) was higher in the vaccinated individuals than in the convalescent patients. A correlation was determined between the severity of the disease and the level of T-cell response. Specific contributions of various groups of antigens to the T-cell response after COVID-19 infection were also determined. Conclusions: the TigraTest^® SARS-CoV-2 kit is a specific and sensitive tool for the assessment of T-cell immunity to the SARS-CoV-2 virus, which can also be used for vaccinated individuals. The kit may be used in clinical practice for comprehensive assessment of immunity to SARS-CoV-2.

INTRODUCTION. Bioanalytical techniques are characterised by greater variability and lower stability than physicochemical methods because live test systems are inherently labile. Since regulatory standards do not establish a unified approach, the selection of system suitability criteria and acceptance criteria for test results is based on validation studies.AIM. This study aimed to validate an analytical procedure for evaluating the biological activity of a medicinal product based on the investigational tocilizumab biosimilar GNR-087 and determine the quantitative limits for system suitability criteria and acceptance criteria for test results.MATERIALS AND METHODS. The biological activity of the investigational tocilizumab biosimilar was assessed by the inhibition of IL-6-induced secreted embryonic alkaline phosphatase (SEAP) expression by HEK-Blue™ IL-6 cells. Statistical processing of the obtained results was performed using Prism 6.0 software.RESULTS. The specificity of the analytical procedure was confirmed by the dose-dependent inhibition of IL-6-induced SEAP expression by cells observed with tocilizumab. The analytical procedure was linear, with a coefficient of determination R²≥ 0.99. The precision of the analytical procedure was satisfactory; its repeat ability varied from 2 to 9%, and its intermediate precision was 14%. The recovery coefficients (Rc) for spiked samples simulating activity levels of 60–140%, including blinded samples, ranged from 80 to 120%. The theoretical values of relative potency (RP) were within the confidence intervals of the mean relative potency estimates, which confirmed the accuracy of the analytical procedure. The validation confirmed the robustness of the analytical procedure to controlled variations, including the use of reporter cells at different passages (with a coefficient of variation for relative potency estimates (CV_RP) of 10%), different IL-6 lots (with a CV_RP of 1%), and different SEAP detection reagent lots (with a CV_RP of 3%); the Rc remained in the range of 80–120% of the nominal RP value.CONCLUSIONS. The analytical procedure for evaluating the biological activity of the investigational tocilizumab biosimilar meets the validation criteria, including accuracy, linearity, precision, specificity, and robustness. The study established system suitability criteria and acceptable limits for biological assay results. This analytical procedure can be used both for routine biological activity control and for demonstrating the biosimilarity of new medicinal products to the original (reference) tocilizumab-based medicinal product.

A proper and representative monitoring of SARS-CoV-2 herd immunity including a long-term health impact on recovered patients and vaccinated individuals is of great importance. For this, a monitoring campaign should assesses both humoral and T-cell immune arms. Upon that, analyzing antigen specific-cell activation and cellular phenotype are informative. We developed a flow cytometry method for detection of intracellular IFN-producing antigen-reactive T cells after exposure of human peripheral blood mononuclear cells (PBMC) to SARS-CoV-2 virus antigens. The method was validated according to the following characteristics: sensitivity, specificity, precision, and robustness. We used positive samples from donors recovered from COVID-19 and negative samples from donors who had no contact with COVID-19 patients and lacking antibodies to SARS-CoV-2. All samples were tested by laboratory methods. Peripheral blood mononuclear cells were isolated from donor blood by centrifugation in a Ficoll density gradient. Specific T cells were stimulated with S-protein as well as N, M, ORF3a, and ORF7a protein peptides to count IFN-producing T cells by flow cytometer. The data were statistically analyzed. The area limited by ROC-curve and false positive rate (AUC) for CD4 and CD8 cells was from 0.97 to 1.00. Precision was considered acceptable because the coefficient of variation for all PBCM did not exceed 20%. Robustness was confirmed for frozen and freshly prepared PBMC samples. The thresholds levels to recognize immune and non-immune samples were defined for CD4-positive T-cells (0.029%) and CD8-positive T-cells (0.0640.068%). Also, acceptance criteria for positive and negative controls were defined. Based on the validation, the suitability of the method Evaluation of antigen-reactive T cells that produce intracellular IFN in response to SARS-CoV-2 virus antigens by flow cytometry was confirmed. The method allows for reliable data that was used to characterize standard control samples for internal quality control of TigraTest SARS-CoV-2 kits.

Background/Objectives: Ocrelizumab is a humanized monoclonal antibody targeting CD20, approved for the treatment of adult patients with relapsing multiple sclerosis (RMS) and primary progressive multiple sclerosis (PPMS). The neutralizing activity of anti-drug antibodies (ADAs), especially neutralizing ADAs (nADAs) activity, should be examined considering that it can alter pharmacokinetic (PK) and pharmacodynamic (PD) profiles, reduce drug efficacy, and lead to life-threatening adverse events. Methods: This article presents data on the development and validation of an assay for neutralizing anti-drug antibodies (nADA) based on ADCC reporter cells for the analysis of patient sera in the context of ocrelizumab clinical studies. Results: Critical steps and conditions to minimize assay variability were identified. The lower limit of detection was 549.6 ng/mL. The cutoff for nonspecific neutralization was determined as 19.7%. The presence of 0.37–3.0 μg/mL ocrelizumab in a biological sample enables the detection of 1.1–10.0 μg/mL polyclonal anti-ocrelizumab idiotype antibodies, respectively. Conclusions: The developed method can be used for immunogenicity studies of medicinal products containing ocrelizumab.

Enzyme replacement therapy (ERT) is one of the most efficient treatments for lysosomal storage diseases. Type 1 Gaucher disease is caused by β-glucocerebrosidase enzyme deficiency, which may be compensated for by intravenous infusions of imiglucerase—a recombinant enzyme. Imiglucerase targets macrophages and enters these cells via interaction with mannose receptors on the cell membrane. Characterisation of internalization of enzymes by target cells is important in the context of the development of new medicines and production of existing ERT medicines. The peritoneal and alveolar macrophages, as well as macrophages of the spleen of small laboratory animals (rats and mice) are widely used in such studies. However, isolation of cells from animal sources raises ethical issues, and therefore continuous mammalian cell lines may offer an attractive alternative.The aim of thestudy:to conduct comparative studies on the internalization of recombinant imiglucerase into mouse peritoneal macrophages and L929 mouse fibroblasts.Materials and methods:CerezymeR batches 7HV0913, C6214H05, 7HV0888 (Genzyme Ltd., UK); Glurazim batches 020416, 011117, 021117 (LLC “IBC “Generium”, Russia). We used peritoneal macrophages obtained from BALB/c mice and L929 mouse fibroblasts. The cells were cultured in DMEM/F12 complete growth medium with 10% fetal bovine serum. The activity of imiglucerase internalized into the cells was evaluated spectrophotometrically by hydrolysis of the artificial substrate—4-methylumbelliferyl-β-Dglucopyranoside.Results:the study compared internalization of recombinant imiglucerase (the active ingredient of CerezymeR and Glurazim) by mouse peritoneal macrophages and L929 mouse fibroblasts. It was demonstrated that the medicines activity in the lysates of peritoneal macrophages is comparable with that in the lysates of L929 mouse fibroblasts. Regardless of the model system, the activity of Glurazim stayed within the acceptable range (80–125%) established for biosimilar products.Conclusions:the experiments proved that L929 mouse fibroblasts could be recommended for assessment of internalization of recombinant imiglucerase.

Представлены результаты сравнительного исследования иммуногенности двухкомпонентной вакцины «Гам-КОВИД-Вак» производства АО «ГЕНЕРИУМ» (Россия) при разных путях введения мышам линии BALB/c (после введения первого или обоих компонентов). Об иммуногенности судили по изменению уровня антигенспецифических антител IgG и IgA в крови и бронхоальвеолярной жидкости, количеству Т-лимфоцитов, вырабатывающих IFN-γ при антигенной стимуляции, количеству антигенспецифических IFN-γ-продуцирующих CD4 и CD8 Т-лимфоцитов. Исследование показало, что интраназальное введение индуцировало развитие мукозального иммунитета, значительно превосходя качественно и количественно эффект внутримышечного введения (по количеству животных с антигенспецифическими антителами IgА и титру антител в сыворотке крови и бронхоальвеолярном лаваже); в то время как внутримышечное несколько превосходило эффект, полученный при интраназальном введении, по влиянию на общее количество продуцирующих IFN-γ CD8-лимфоцитов.