MAINTAINING IMMUNOLOGICAL MEMORY TO THE SARS-CoV-2 VIRUS DURING COVID-19 PANDEMIC.

The question on the duration and effectiveness of post-infection vs post-vaccination SARS-CoV-2 immunity remains in the focus of numerous studies. The aim of the work was to examine the duration of maintained post-infection and post-vaccination SARS-CoV-2 immunity as well as formation of hybrid (vaccination after infection) and breakthrough (repeated disease or disease after vaccination) immunity in the context of an ongoing COVID-19 pandemic. 107 adults with mild or moderate COVID-19 3-18 months after the disease and 30 subjects vaccinated twice with the Sputnik V vaccine were examined 1-6 times. Antibodies against SARS-CoV-2 virus were determined by ELISA on the "SARSCoV-2-IgG quantitative-ELISA-BEST" test systems. The antibody avidity was measured by additional incubation with and without denaturing solution. Mononuclear cells were isolated from blood by gradient centrifugation, incubated with and without coronavirus S-protein for 20 hours, stained with fluorescently labeled antibodies, and the percentage of CD8highCD107a+ was counted using FACSCanto II cytometer. It was shown that in the group of convalescent and vaccinated subjects, the level of virus-specific antibodies decreased more deeply in individuals with initially high humoral response, but 9 months later the decrease slowed down and reached a plateau. The antibody avidity rose up to 50% and persisted for 18 months. Cellular immunity in recovered patients did not change for 1.5 years, while in vaccinated patients it gradually decreased 6 months later, but remained at detectable level. After revaccination, a significant increase in the level of antibodies, avidity up to 67.6% and cellular immunity returned to the initial level were noted. Hybrid immunity turned out to be significantly higher than post-infection and post-vaccination immunity. The level of antibodies increased to 1218.2 BAU/ml, avidity - to 69.85%, and cellular immunity - to 9.94%. Breakthrough immunity was significantly higher than that after the first disease. The level of antibodies rose to 1601 BAU/ml, avidity - up to 81.6%, cellular immunity - up to 13.71%. Using dynamic observation of four COVID-19 convalescents, it has been shown that in the context of the ongoing pandemic and active coronavirus mutation, natural boosting occurs both asymptomatically and as a result of a mild re-infection, which prevents disappearance of SARS-CoV-2 humoral and cellular immunity.Copyright © 2023 Saint Petersburg Pasteur Institute. All rights reserved.

Comparison of the Humoral and Cellular Immunity in Covid-19 Convalescents

The SARS-CoV-2 virus caused the COVID-19 pandemic is related to the SARS-CoV-1 and MERS coronaviruses, which were resulted in 2003 and 2012 epidemics. Antibodies in patients with COVID-19 emerge 7-14 days after the onset of symptoms and gradually increase. Because the COVID-19 pandemic is still in progress, it is hard to say how long the immunological memory to the SARS-CoV-2 virus may be retained. The aim of this study was to study a ratio between humoral and cellular immunity against the SARS-CoV-2 S protein in COVID-19 convalescents. There were enrolled 60 adults with mild to moderate COVID-19 2 to 12 months prior to the examination. The control group consisted of 15 adults without COVID-19 or unvaccinated. Specific antibodies to the SARS-CoV-2 virus were determined by ELISA with the SARS-CoV-2-IgG-ELISA-BEST kit. To determine the specific IgG and IgA subclasses, the anti-IgG conjugate from the kit was replaced with a conjugate against the IgG subclasses and IgA. Additional incubation with or without denaturing urea solution was used to determine the avidity of antibodies. Peripheral blood mononuclear cells were isolated by gradient centrifugation, incubated with or without coronavirus S antigen for 20 hours, stained by fluorescently labeled antibodies, and the percentage of CD8(high)CD107a cells was assessed on flow cytometer BD FACSCanto II. In the control group, neither humoral nor cellular immunity against the SARS-CoV-2 S protein was found. In the group of convalescents, the level of IgG antibodies against the SARS-CoV-2 S protein varies greatly not being strictly associated with the disease duration, with 57% and 43% of COVID-19 patients having high vs. low level of humoral response, respectively. A correlation between level of specific IgG and IgA was r = 0.43. The avidity of antibodies increased over time in convalescents comprising 49.9% at 6-12 months afterwards. No virus-specific IgG2 and IgG4 subclasses were detected, and the percentage of IgG1 increased over time comprising 100% 6-12 months after recovery. 50% of the subjects examined had high cellular immunity, no correlations with the level of humoral immunity were found. We identified 4 combinations of humoral and cellular immunity against the SARS- CoV-2 S protein: high humoral and cellular, low humoral and cellular, high humoral and low cellular, and vice versa, low humoral and high cellular immunity.

COMPARISON OF THE HUMORAL AND CELLULAR IMMUNITY IN COVID-19 CONVALESCENTS

The SARS-CoV-2 virus caused the COVID-19 pandemic is related to the SARS-CoV-1 and MERS coronaviruses, which were resulted in 2003 and 2012 epidemics. Antibodies in patients with COVID-19 emerge 7–14 days after the onset of symptoms and gradually increase. Because the COVID-19 pandemic is still in progress, it is hard to say how long the immunological memory to the SARS-CoV-2 virus may be retained. The aim of this study was to study a ratio between humoral and cellular immunity against the SARS-CoV-2 S protein in COVID-19 convalescents. There were enrolled 60 adults with mild to moderate COVID-19 2 to 12 months prior to the examination. The control group consisted of 15 adults without COVID-19 or unvaccinated. Specific antibodies to the SARS-CoV-2 virus were determined by ELISA with the SARS-CoV-2-IgG-ELISA-BEST kit. To determine the specific IgG and IgA subclasses, the anti-IgG conjugate from the kit was replaced with a conjugate against the IgG subclasses and IgA. Additional incubation with or without denaturing urea solution was used to determine the avidity of antibodies. Peripheral blood mononuclear cells were isolated by gradient centrifugation, incubated with or without coronavirus S antigen for 20 hours, stained by fluorescently labeled antibodies, and the percentage of CD8highCD107a cells was assessed on flow cytometer BD FACSCanto II. In the control group, neither humoral nor cellular immunity against the SARS-CoV-2 S protein was found. In the group of convalescents, the level of IgG antibodies against the SARS-CoV-2 S protein varies greatly not being strictly associated with the disease duration, with 57% and 43% of COVID-19 patients having high vs. low level of humoral response, respectively. A correlation between level of specific IgG and IgA was r = 0.43. The avidity of antibodies increased over time in convalescents comprising 49.9% at 6–12 months afterwards. No virus-specific IgG2 and IgG4 subclasses were detected, and the percentage of IgG1 increased over time comprising 100% 6–12 months after recovery. 50% of the subjects examined had high cellular immunity, no correlations with the level of humoral immunity were found. We identified 4 combinations of humoral and cellular immunity against the SARS-CoV-2 S protein: high humoral and cellular, low humoral and cellular, high humoral and low cellular, and vice versa, low humoral and high cellular immunity.

ASSESSMENT of SEROLOGICAL TESTS for ANTIBODIES to DIFFERENT ANTIGENS of the SARS-CoV-2 VIRUS: COMPARISON of SIX IMMUNOASSAYS

The new coronavirus SARS-CoV-2 has become a global challenge to medicine and, in particular, laboratory diagnostics. The study of the antibodies' level to SARS-CoV-2 can be used as a confirmation test in the diagnosis of a disease, but it becomes of paramount importance in assessing population immunity resulting from a disease or vaccination, as well as in selection of convalescent plasma donors. The kits developed in our country and abroad for detecting antibodies to the SARS-CoV-2 virus differ both in the methods of testing and in the used coronavirus antigens to which the antibodies are directed. The aim of this study was to compare the diagnostic sensitivity and specificity of five kits for the detection of IgG antibodies to the SARS-CoV-2 virus, based on different diagnostic methods. Serum samples from 137 COVID-19 convalescents and 166 donors of blood and its components were examined. The control group consisted of 50 blood sera collected at the beginning of 2019 and 19 sera collected in 2018 (before the advent of the SARS-CoV-2 virus) and stored at -70 °C. Testing was carried out in analytical systems: rapid test “COVID-19 IgM/IgG Rapid Test (Colloidal Gold)” (China), on an automatic immunochemical analyzer Abbott Architect™ i2000 and kit “SARS-CoV-2-IgG” (Abbot, Chicago, IL USA), by the chemiluminescence method using an automatic analyzer of the CL series and kits of the “Mindray” company (China) “SARS-CoV-2 IgM” and “SARS-CoV-2 IgG” and by the enzyme immunoassay method on the kits of the companies “Diagnostic Systems” Ltd (Russia, Nizhny Novgorod) “DS-IFA-ANTI-SARS-CoV-2-G”, “Xema” Ltd (Federal State Budgetary Institution “National Medical Research Center of Hematology” of the Ministry of Health of Russia) “SARS-CoV-2-IgG-IFA” and “Vector-Best” CJSC (Russia, Novosibirsk)” SARS-COV-2-IgM-IFA-BEST” and “SARS-COV-2-IgG-IFABEST”. When comparing the results of testing 137 plasma samples on the Vector-Best and Mindray kits for IgG antibodies, 127 samples were positive, 7 samples were negative on both kits, the discrepancy was 2.2%. In the study of IgM antibodies, 32.1% were positive, and 52.6% were negative in both kits. The discrepancy rate was 15.3%. Out of 166 samples, 1 serum (0.6%) was negative in 5 kits. On the Mindray kit, IgG antibodies to the antigens of the SARS-CoV-2 virus were detected in 165 samples (99.4%), on Vector-Best - in 164 sera (98.8%), on Diagnostic systems - in 151 (90.96%), on Xema - in 154 (92.8%), and on Abbott - in 155 samples (93.4%). At the same time, 135 (81.33%) samples were positive in all kits, while 30 samples had discordant results (18.07%), and in 9 sera, specific IgG was not detected in 2 or more kits. ROC analysis revealed a high diagnostic value of all tested kits (AUC from 0.908 to 0.998), which indicates a high quality of the separation model of positive and negative samples (p < 0.001). With the cut-off set by the manufacturers, the sensitivity and specificity ranged from 82.8% and 93.3% for the Diagnostic Systems kit to 99.4% and 95.8% for the Vector-Best kit. The calculated correlation coefficients were higher between kits with a similar composition of the antigen used in the kits;therefore, it is better to monitor the dynamics of antibodies by diagnostic kits from the same manufacturer. © 2021 Russian Association of Allergologists and Clinical Immunologists, St. Petersburg Regional Branch (SPb RAACI). All rights reserved.

Gating strategy for plasmablast enumeration after hepatitis b vaccination

B cell stimulation develops upon vaccination, thus causing occurrence of activated B cells (plasmoblasts) in bloodstream. Similar cells are also observed in some viral infections. The contents of plasmablasts may be a marker of successful vaccination, or a diagnostic feature of ongoing infection. The plasmablasts are normally represented by a small cell subpopulation which is not easy to detect. A study was performed with 15 healthy volunteers who were subjected to a single immunization with a recombinant vaccine against hepatitis B virus. To identify the plasmablasts, we have used labeled antibodies prepared in our laboratory. These reagents were previously validated for counting the plasmablasts. Different gating strategies for plasmablast gating have been compared. Upon staining of lymphocytes from immunized volunteers, we observed a distinct cluster of plasmablasts with CD27++CD38++ phenotype using the following antibody set: CD19-PE, CD3/CD14/CD16-FITC, CD27-PC5.5 and CD38-PC7. Inclusion of a CD20-FITC antibody into the panel caused an increase of CD27++CD38++ plasmablast ratio among CD19+ lymphocytes to > 60%. Upon substitution of CD38 antibody by anti-CD71, a distinct plasmablast cluster was again revealed, which contained ca. 5 per cent B cells. Two strategies for the plasmablast gating using the CD27/ CD38 and CD27/CD71 combinations were compared in dynamics with lymphocyte samples from a single vaccinated volunteer. When applying the CD27/CD38 combination, a sharp and pronounced plasmablast peak was registered on day 7 post-vaccination. With CD27/CD71 combination, the peak was extended between day 7 and day 14 following immunization. Hence, time kinetics of the CD27+CD71+ population proved to be different from occurrence of classic plasmablasts with CD27++CD38++ phenotype. This finding suggests that the CD27++CD71+ population contains both plasmablasts and other types of activated B cells. A minor HBV surface antigen was prepared and labeled with phycoerythrin (HBsAg-PE), thus allowing to quantify the antigen-specific plasmablasts. The results of HBsAg-PE-based detection of antigen-specific cells were in compliance with the data obtained by ELISpot technique. At the present time, we use the original plasmablast gating technique for detection of activated B cells in SARS-CoV-2 infection. At the next step, this technique will be applied to sorting of antigen-specific B cells, thus permitting sequencing of Ig genes and design of novel human antibodies against viral antigens. © 2020, SPb RAACI.