Expansion of SARS-CoV-2–Specific Antibody-Secreting Cells and Generation of Neutralizing Antibodies in Hospitalized COVID-19 Patients
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Abstract
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in late 2019 and has since become a global pandemic. Pathogen-specific Abs are typically a major predictor of protective immunity, yet human B cell and Ab responses during COVID-19 are not fully understood. In this study, we analyzed Ab-secreting cell and Ab responses in 20 hospitalized COVID-19 patients. The patients exhibited typical symptoms of COVID-19 and presented with reduced lymphocyte numbers and increased T cell and B cell activation. Importantly, we detected an expansion of SARS-CoV-2 nucleocapsid protein–specific Ab-secreting cells in all 20 COVID-19 patients using a multicolor FluoroSpot Assay. Out of the 20 patients, 16 had developed SARS-CoV-2–neutralizing Abs by the time of inclusion in the study. SARS-CoV-2–specific IgA, IgG, and IgM Ab levels positively correlated with SARS-CoV-2–neutralizing Ab titers, suggesting that SARS-CoV-2–specific Ab levels may reflect the titers of neutralizing Abs in COVID-19 patients during the acute phase of infection. Last, we showed that IL-6 and C-reactive protein serum concentrations were higher in patients who were hospitalized for longer, supporting the recent observations that IL-6 and C-reactive protein could be used as markers for COVID-19 severity. Altogether, this study constitutes a detailed description of clinical and immunological parameters in 20 COVID-19 patients, with a focus on B cell and Ab responses, and describes tools to study immune responses to SARS-CoV-2 infection and vaccination.
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SciScore for 10.1101/2020.05.28.118729: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Ethics statement: The study was approved by the Regional Ethical Review Board in Stockholm, Sweden and by the Swedish Ethical Review Authority.
Consent: All COVID-19 patients and healthy controls included in this study provided a written informed consent for participation.Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable Study subjects and sampling of peripheral blood: Peripheral blood samples were collected from 20 adult COVID-19 patients hospitalized in April 2020 at the Karolinska University Hospital in Stockholm, Sweden (5 females and 15 males; age range between 34 and 67 years; median age 53 years) … SciScore for 10.1101/2020.05.28.118729: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Ethics statement: The study was approved by the Regional Ethical Review Board in Stockholm, Sweden and by the Swedish Ethical Review Authority.
Consent: All COVID-19 patients and healthy controls included in this study provided a written informed consent for participation.Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable Study subjects and sampling of peripheral blood: Peripheral blood samples were collected from 20 adult COVID-19 patients hospitalized in April 2020 at the Karolinska University Hospital in Stockholm, Sweden (5 females and 15 males; age range between 34 and 67 years; median age 53 years) (Table I). Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The following monoclonal antibody conjugates were used for cell surface staining: anti-CD8-Qdot605 (3B5) (Thermo Fisher Scientific), anti-CD19-BUV395 (SJ25C1), anti-CD14-V500 (MφP9), anti-CD4 -BUV737 (RPA-T4) (all from BD Biosciences), anti-CD123-BV510 (6H6), anti-CD27-BV650 (O323), anti-CD20-FITC (2H7), anti-CD38-BV421 (HB-7), anti-IgD-PE-Cy7 (IA6-2), anti-IgM-BV785 (MHM-88) (all from BioLegend), anti-CD3-PE-Cy5 (UCHT1), anti-CD56-ECD (N901) (all from Beckman Coulter), and anti-IgA-APC (REA1014) (Miltenyi). anti-CD8-Qdot605suggested: Noneanti-CD19-BUV395suggested: Noneanti-CD14-V500suggested: Noneanti-CD4suggested: Noneanti-CD123-BV510suggested: Noneanti-CD27-BV650suggested: Noneanti-CD20-FITCsuggested: (MBL International Cat# K0170-4, RRID:AB_590955)anti-CD38-BV421suggested: Noneanti-IgD-PE-Cy7suggested: Noneanti-IgM-BV785suggested: Noneanti-CD3-PE-Cy5suggested: Noneanti-CD56-ECD (N901suggested: Noneanti-IgA-APC (REA1014suggested: NoneThe following monoclonal antibody conjugates were used for intracellular staining: anti-IgG-PE (HP6017) (BioLegend) and anti-Ki-67-AF700 (B56) (BD Biosciences). anti-IgG-PEsuggested: (Miltenyi Biotec Cat# 130-099-201, RRID:AB_2661448)HP6017suggested: Noneanti-Ki-67-AF700suggested: NoneB56suggested: NoneFluoroSpot assay for antibody-secreting cells: The number of SARS-CoV-2 nucleocapsid (N) protein-specific IgA, IgG and IgM antibody-secreting cells (ASCs), as well as the total number of IgA-, IgG- and IgM-ASCs in freshly isolated PBMCs were measured using a multicolor B cell FluoroSpot kit with modifications (Mabtech). IgA, IgGsuggested: NoneBriefly, ethanol-activated IPFL membrane plates were coated overnight with either: (i) anti-IgG, anti-IgA, and anti-IgM capture antibodies (15µg/mL of each) for the detection of all ASCs, or (ii) SARS-CoV-2 N protein (10 µg/mL) for the detection of SARS-CoV-2-specific ASCs. anti-IgGsuggested: Noneanti-IgMsuggested: NonePlates were then incubated at 37°C in 5% CO2 for 20 hours and then developed with anti-human IgG-550 (yellow fluorescence), anti-human IgA-490 (green fluorescence) and anti-human IgM-640 (red fluorescence) secondary detection antibodies (diluted 1:500 each) (all antibodies from Mabtech). anti-human IgG-550suggested: Noneanti-human IgA-490suggested: Noneanti-human IgM-640suggested: NoneFor analysis of total SARS-CoV-2 IgG antibody titers, serum samples were serially diluted from 1:20 to 1:5120. 25 µL of diluted serum was then added to fixed cells and incubated at 37°C for 30 min, after which the slides were washed in NaCl for 30 min. SARS-CoV-2 IgGsuggested: NoneBound SARS-CoV-2 IgG antibodies were then detected by incubating for 30 min at 37°C with a secondary AF488-conjugated AffiniPure goat anti-human IgG antibody (Jackson Immunoresearch), diluted 1:200 in 0.1% Evan’s Blue. Bound SARS-CoV-2 IgG antibodiessuggested: Noneanti-human IgGsuggested: NoneELISAs: SARS-CoV-2 specific IgG and IgA antibodies in serum were detected using anti-SARS-CoV-2 ELISA kits (both from Euroimmun), according to the manufacturer’s instructions. IgAsuggested: Noneanti-SARS-CoV-2suggested: NoneSARS-CoV-2 specific IgM antibodies were detected using EDI Novel Coronavirus COVID-19 IgM ELISA kit (Epitope Diagnostics), according to the manufacturer’s instructions. specific IgMsuggested: NoneExperimental Models: Cell Lines Sentences Resources Immunofluorescence assay (IFA) for IgG against SARS-CoV-2: Vero E6 cells were infected with SARS-CoV-2 (isolate SARS-CoV-2/human/SWE/01/2020, accession number MT093571) for 24 hours, trypsinized and mixed with uninfected Vero E6 cells, and then seeded on microscope slides. Vero E6suggested: NoneSoftware and Algorithms Sentences Resources Finally, samples were incubated in a 2% formaldehyde solution (Polysciences) for 2 h, washed and resuspended in flow cytometry buffer, and data subsequently acquired on a BD LSRFortessa flow cytometer equipped with 355, 405, 488, 561, and 639 nm lasers and BD FACSDiva Software (BD Biosciences). BD FACSDivasuggested: (BD FACSDiva Software, RRID:SCR_001456)The following monoclonal antibody conjugates were used for cell surface staining: anti-CD8-Qdot605 (3B5) (Thermo Fisher Scientific), anti-CD19-BUV395 (SJ25C1), anti-CD14-V500 (MφP9), anti-CD4 -BUV737 (RPA-T4) (all from BD Biosciences), anti-CD123-BV510 (6H6), anti-CD27-BV650 (O323), anti-CD20-FITC (2H7), anti-CD38-BV421 (HB-7), anti-IgD-PE-Cy7 (IA6-2), anti-IgM-BV785 (MHM-88) (all from BioLegend), anti-CD3-PE-Cy5 (UCHT1), anti-CD56-ECD (N901) (all from Beckman Coulter), and anti-IgA-APC (REA1014) (Miltenyi). BD Biosciencessuggested: (BD Biosciences, RRID:SCR_013311)Statistics and data analysis: Statistical analyses were performed using GraphPad Prism software 7.0 for MacOSX (GraphPad Software). GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)FlowJo software version 10.5.3 (Tree Star) was used to analyze all flow cytometry data. FlowJosuggested: (FlowJo, RRID:SCR_008520)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- No funding statement was detected.
- No protocol registration statement was detected.
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