T-Cell Immunity in COVID-19-Recovered Individuals and Individuals Vaccinated with the Combined Vector Vaccine Gam-COVID-Vac.

The COVID-19 pandemic has required extensive research on the new coronavirus SARS-CoV-2 and the creation of new highly effective vaccines. The presence of T-cells in the body that respond to virus antigens suggests adequate antiviral immunity. We investigated T-cell immunity in individuals who recovered from mild and moderate COVID-19 and in individuals vaccinated with the Gam-COVID-Vac combined vector vaccine. The ELISPOT method was used to determine the number of T-cells responding with IFN-γ synthesis to stimulation by peptides containing epitopes of the S-protein or N-, M-, ORF3, and ORF7 proteins, using peripheral blood mononuclear cells (PBMCs). At the same time, the multiplex method was used to determine the accumulation of IFN-γ and other cytokines in the culture medium. According to the data obtained, the proportion of positive conclusions about the T-cell immune response to SARS-CoV-2 antigens in control, recovered, and vaccinated individuals was 12%, 70%, and 52%, respectively. At the same time, more than half of the vaccinated individuals with a T-cell response were sensitized to the antigens of N-, M-, ORF3, and ORF7 proteins not produced by Gam-COVID-Vac, indicating a high likelihood of asymptomatic SARS-CoV-2 infection. Increased IFN-γ release by single sensitized T-cells in response to specific stimulation in recovered and vaccinated individuals did not result in the accumulation of this and other cytokines in the culture medium. These findings suggest a balance between cytokine production and utilization by immunocompetent cells as a prerequisite for providing a controlled cytokine signal and avoiding a "cytokine storm".

Von Willebrand Factor and ADAMTS-13 Are Associated with the Severity of COVID-19 Disease.

Coagulopathy in COVID-19 patients is presumably based on systemic hypercoagulation with the inflammatory response. As a result of endothelial dysfunction, tissue factor and von Willebrand factor (vWF) are released into the blood stream, which leads to prothrombinase activation. The vWF/ADAMTS-13 ratio can be used for monitoring the severity of the disease. This observational prospective study included 141 patients with COVID-19. In patients with mild COVID-19 (group 1), the assessment was performed on the 3rd-7th day of illness (point 1) and 14-28 days after recovery (point 2). In patients with moderate (groups 2) and severe (group 3) COVID-19, the assessment was performed during hospitalization (point 1) and after 14 days (point 2). The vWF:RCo/ADAMTS-13:activity (point 1), vWF/ADAMTS-13 (point 2) and vWF:RCo/ADAMTS-13:activity (point 2) ratios were significantly higher in patients with moderate and severe COVID-19. Moreover, in these patients, both ratios increased after recovery (point 2), which is a negative prognostic factor of thrombotic complications. Thus, COVID-19 is characterized by a decrease in the concentration and activity of ADAMTS-13 metalloproteinase, especially in patients with the severe form of COVID-19. A decrease in ADAMTS-13 activity results in an increase in vWF concentration and activity so the ratio of vWF to ADAMTS-13 changes significantly.

SARS-CoV-2 Peptide Bioconjugates Designed for Antibody Diagnostics.

In the near future, the increase in the number of required tests for COVID-19 antibodies is expected to be many hundreds of millions. Obviously, this will be done using a variety of analytical methods and using different antigens, including peptides. In this work, we compare three method variations for detecting specific immunoglobulins directed against peptides of approximately 15-aa of the SARS-CoV-2 spike protein. These linear peptide epitopes were selected using antigenicity algorithms, and were synthesized with an additional terminal cysteine residue for their bioconjugation. In two of the methods, constructs were prepared where the peptide (F, function) is attached to a negatively charged hydrophilic spacer (S) linked to a dioleoylphosphatidyl ethanolamine residue (L, lipid) to create a function-spacer-lipid construct (FSL). These FSLs were easily and controllably incorporated into erythrocytes for serologic testing or in a lipid bilayer deposited on a polystyrene microplate for use in an enzyme immunoassays (EIA). The third method, also an EIA, used polyacrylamide conjugated peptides (peptide-PAA) prepared by controlled condensation of the cysteine residue of the peptide with the maleimide-derived PAA polymer which were immobilized on polystyrene microplates by physisorption of the polymer. In this work, we describe the synthesis of the PAA and FSL peptide bioconjugates, design of test systems, and comparison of the bioassays results, and discuss potential reasons for higher performance of the FSL conjugates, particularly in the erythrocyte-based serologic assay.

Detection of antibodies to SARS-CoV-2 in healthcare professionals of the National Center during the COVID-19 pandemic (April-June 2020)

Objective. To identify IgG antibodies to SARS-CoV-2 in healthcare professionals of the National Medical Research Center for Obstetrics, Gynecology and Perinatology during the quarantine from April to June 2020. Materials and methods. The study included 1589 healthcare workers: 1293 professionals of ‘green zone’ and 926 medical staff of‘red zone’. IgG antibodies to SARS-CoV-2 in blood serum were determined using SARS-CoV-2-IgG- ELISA kits (National Hematology Research Center, Russia). SARS-CoV-2 RNA was extracted from nasopharyngeal swabs using the kit PROBA-NK(DNA-technology, LLC, Russia). The virus was identified by RT-PCR using SARS-CoV-2/SARS-CoVMultiplex REAL-TIME PCR Detection Kit (DNA-technology, LLC, Russia). Results. IgG antibodies to SARS-CoV-2 were detected in 141 healthcare workers (8.9%), controversial results were revealed in 2professionals, and 1445 (90.9%) workers had no antibodies, including 46 (3.2%) people who had the clinical symptoms of acute respiratory viral disease (ARVI) and identified SARS-CoV-2 RNA. Among healthcare workers with antibodies, the clinical symptoms of ARVI were revealed in 129 (91.5%) workers, they were also detected SARS- CoV-2 RNA;23 (17.8%) people had clinical symptoms of ARVI but SaRS- CoV-2 RNA was not extracted;12 (8,5%) workers had neither clinical symptoms of ARVI nor detected SARS- CoV-2 RNA. Conclusion. The presence of IgG antibodies and the absence of SARS-CoV-2 RNA in the nasopharyngeal swab as well as clinical symptoms of the disease may be suggestive of the fact that the worker had this disease;the presence of IgG antibodies and the absence of SARS- CoV-2 RNA as well as clinical symptoms of the disease may be suggestive of the fact that the worker has this disease. Цель. Определение антител класса IgG к SARS-CoV-2 у сотрудников ФГБУ «НМИЦ АГП им. В.И. Кулакова» МЗ РФ в период карантинных мероприятий в апреле-июне 2020 г. Материалы и методы. Обследовано 1589 сотрудников: 1293 сотрудника «зеленой» зоны и 296 сотрудников «красной» зоны. Определение антител к SARS- CoV-2 класса IgG в сыворотке крови осуществляли наборами реагентов «SARS-CoV-2-IgG-ИФА» («НМИЦ гематологии» МЗ РФ). Выделение РНК SARS-CoV-2 из соскоба из ротоглотки проводили с использованием реагентов ПРОБА-НК (ООО «НПО ДНК-Технология», Россия). Вирус идентифицировали методом РТ-ПЦР с помощью «Набора реагентов для выявления РНКкоронавирусов SARS-CoV-2 и подобных SARS-CoVметодом обратной транскрипции и полимеразной цепной реакции в режиме реального времени (SARS-CoV-2/ SARS-CoV)» (ООО нПо «ДНК-Технология», Россия). Результаты. Антитела к SARS-CoV-2 класса IgG были выявлены у 141 сотрудника (8,9%), у 2 был сомнительный результат, у 1445 (90,9%) антитела не были выявлены, но среди них у 46 (3,2%) человек наблюдались клинические симптомы ОРВИ и была выявлена РНК SARS-CoV-2. Среди сотрудников с наличием антител у 129 (91,5%) наблюдались клинические симптомы ОРВИ и была выявлена РНК SARS-CoV-2;у 23 (17,8%) сотрудников наблюдались клинические симптомы ОРВИ без выявления РНК SARS- CoV-2;у 12 (8,5%) отсутствовали клинические симптомы ОРВИ и не была выявлена РНК SARS-CoV-2. Заключение. Наличие антител класса IgG при отсутствии РНК SARS- CoV-2 в соскобе из ротоглотки и клинических симптомов заболевания свидетельствует о перенесенном заболевании;наличие антител класса IgG при отсутствии РНК SARS- CoV-2 и проявлении клинических симптомов заболевания свидетельствует о текущем заболевании.