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Background: Serum virus-neutralization (VNT)capacity is an important parameter of immunological response in COVID-19 recovered individuals and it can also be used to predict the effectiveness of therapy with COVID-19 convalescent plasma. The most commonly used tests to assess virus-neutralization are those that use native SARS-CoV- 2 (cVNT), SARS-CoV- 2 spike pseudotyped lentivirus-like particles (pVNT), and also inhibition of recombinant RBD binding to ACE2 receptor in vitro (surrogate VNT, sVNT). The aim of this study was to determine the interchangeability of different approaches in the evaluation of the virus-neutralization activity of convalescent serum samples. Method(s): Serum samples (n = 111) were collected 10-36 days after recovery between May and September 2020. The SARS-CoV- 2 strain PMVL-12/ 2020 was used in cVNT at 100 doses of TCID50. In pVNT SARS-CoV- 2 pseudotyped HIV-1 based virus-like particles with GFP reporter gene were used. sVNT was performed with a kit from Xema Co., Russia. Result(s): A very strong correlation was observed between cVNT and pVNT results (Spearman's r = 0.841). The correlation of cVNT and pVNT with the sVNT was only moderate (r = 0.674 and 0.696, respectively). This is consistent with the fact that sVNT detects only RBD blocking antibodies, which are the main but not the only inhibitors of viral infection. Serum samples were also tested for RBD-specific IgG, IgM, IgA antibodies by ELISA. A good correlation was found between the cVNT, pVNT, sVNT results and the RBD-specific IgG (r = 0.669, 0.620, and 0.643, respectively), that confirms a crucial role of specific IgG antibodies in virus-neutralization. RBD-specific IgA showed a moderate correlation with the neutralization capacity of sera from recovered individuals (r = 0.563, 0.583, 0.544). Correlation of approximately the same level was observed between cVNT, pVNT, and RBD-specific IgM (r = 0.663, 0.615), but not sVNT and RBD-specific IgM (r = 0.395). Conclusion(s): This study demonstrated the possibility of using the safe and relatively simple pseudotyped virus-neutralization test instead of cVNT to assess the sera virus-neutralizing capacity. sVNT may be efficiently used in screening studies.
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The parameters of the humoral response are an important immunological characteristic of donors who recovered from COVID-19 and vaccinated individuals. Analysis of the level of virus-binding antibodies has become widespread. The most accurate predictor of effective immune protection against symptomatic SARS-CoV-2 infection is the activity of virus-neutralizing antibodies. We determined virus-neutralizing activities in plasma samples of individuals (n = 111) who had COVID-19 from April to September 2020. Three independent methods were used: conventional with live virus, with virus-like particles pseudotyped with spike protein, and a surrogate virus-neutralization test (cVNT, pVNT and sVNT, respectively). For comparison, the levels of IgG, IgA and IgM antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein were also evaluated. The levels of virus-binding as well as virus-neutralizing antibodies in cVNT and pVNT showed high heterogeneity. A comparison of cVNT and pVNT results showed a high correlation, sVNT results also correlated well with both cVNt and pVNT. To the greatest extent, the level of IgG antibodies correlated with the results of cVNT, pVNT and sVNT. These results can be used in the selection of plasmas that are best suited for transfusion and treatment of acute COVID-19. In addition, data on the virus-neutralizing activity of plasma are important for the selection of potential donors, for the isolation of SARS-CoV-2-specific B-lymphocytes, in order to further generate monoclonal virus-neutralizing antibodies.
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Introduction. The determination of antibodies against the Spike (S) protein of the novel coronavirus is widely used to confirm current or past infection with SARS-CoV-2, and as an indicator of the effectiveness of vaccination against COVID-19. The most common method for detecting anti-S-antibodies is enzyme-linked immunosorbent assay (ELISA), which uses a recombinant S-protein. Immunofluorescence followed by flow cytometry provides an alternative approach to detect anti-S-antibodies, where a protein in the native transmembrane conformation is used as the S-antigen. The aim of the study was to develop a method for determining anti-S-antibodies using flow cytometry, and to select the most appropriate method for processing experimental data. Material and methods. The study involved 22 volunteers (7 men and 15 women aged 25 to 70 years, median 48). All volunteers were vaccinated with two doses of the <<Sputnik V>> vaccine between January and February 2021. Donor sera samples were collected before vaccination with <<Sputnik V>> and 3 months after vaccination. 5 volunteers had already had a mild form of COVID-19 before the time of vaccination. The remaining 17 volunteers did not encounter the SARS-CoV-2. Antibodies against S-protein were determined by immunofluorescence with registration on a flow cytometer. HEK293 cells were transiently transfected with a plasmid encoding the wild type S-protein which was used as target. Transfection was performed by the calcium phosphate method. Cells were incubated with serially diluted sera and then stained with anti-IgG-PE and anti-IgM-FITC secondary antibodies. The fluorescence level was measured using a flow cytometer. As a measurement result, the mean fluorescence intensity (MFI) obtained at 1:18 serum dilution, or the area under the titration curve (area under curve, AUC) was used. Anti-RBD-antibodies were determined using enzyme immunoassay, and virus-neutralizing activity using pseudotyped or surrogate virus-neutralization analysis (pVNA and sVNA). Results. Using the developed method, the formation of anti-S antibodies of the IgG and IgM isotypes was shown 3 months after immunization with the <<Sputnik V>> vaccine. In a simplified version of the method, the relative concentration of antibodies was determined at a single dilution of the test serum by measuring the mean fluorescence intensity (MFI) of the target cells. More reliable results were obtained by construction the titration curve and calculating the area under the curve (AUC). The results thus obtained correlated well with the detection of anti-RBD antibodies by ELISA, as well as with virus neutralization data in pseudotyped and surrogate assays. Conclusion. Flow cytometry is a convenient method for the simultaneous determination of anti-S antibodies of IgG and IgM isotypes in human serum. The advantages of the method include the fact that the S-protein is presented in a native transmembrane conformation. After minor modification, the established method can be used to determine the level of anti-S-antibodies against mutant variants of SARS-CoV-2. Copyright © 2022 Meditsina Publishers. All rights reserved.
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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.