High‐throughput detection of antibodies targeting the SARS‐CoV ‐2 Spike in longitudinal convalescent plasma samples
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Abstract
Background
The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) virus is the cause of the ongoing coronavirus disease 2019 (COVID‐19) pandemic, infecting millions of people and causing more than two million deaths. The SARS‐CoV‐2 Spike glycoproteins mediate viral entry and represent the main target for antibody responses. Humoral responses were shown to be important for preventing and controlling infection by coronaviruses. A promising approach to reduce the severity of COVID‐19 is the transfusion of convalescent plasma. However, longitudinal studies revealed that the level of antibodies targeting the receptor‐binding domain (RBD) of the SARS‐CoV‐2 Spike declines rapidly after the resolution of the infection.
Study Design and Methods
To extend this observation beyond the RBD domain, we performed a longitudinal analysis of the persistence of antibodies targeting the full‐length SARS‐CoV‐2 Spike in the plasma from 15 convalescent donors. We generated a 293T cell line constitutively expressing the SARS‐CoV‐2 Spike and used it to develop a high‐throughput flow cytometry‐based assay to detect SARS‐CoV‐2 Spike‐specific antibodies in the plasma of convalescent donors.
Results and Conclusion
We found that the level of antibodies targeting the full‐length SARS‐CoV‐2 Spike declines gradually after the resolution of the infection. This decline was not related to the number of donations but strongly correlated with the decline of RBD‐specific antibodies and the number of days post‐symptom onset. These findings help to better understand the decline of humoral responses against the SARS‐CoV‐2 Spike and provide important information on when to collect plasma after recovery from active infection for convalescent plasma transfusion.
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SciScore for 10.1101/2020.10.20.346783: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Consent: Males and females with no history of pregnancy meeting the above criteria were invited to donate plasma, after informed consent. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable Males and females with no history of pregnancy meeting the above criteria were invited to donate plasma, after informed consent. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources AlexaFluor-647-conjugated goat anti-human IgG (H+L) Abs (Invitrogen) were used as secondary antibodies. anti-human IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources 293T cells were transfected with 10 μg … SciScore for 10.1101/2020.10.20.346783: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Consent: Males and females with no history of pregnancy meeting the above criteria were invited to donate plasma, after informed consent. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable Males and females with no history of pregnancy meeting the above criteria were invited to donate plasma, after informed consent. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources AlexaFluor-647-conjugated goat anti-human IgG (H+L) Abs (Invitrogen) were used as secondary antibodies. anti-human IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources 293T cells were transfected with 10 μg of Spike expressor and 2 μg of a green fluorescent protein (GFP) expressor (pIRES-GFP) for 2×106 293T cells using the standard calcium phosphate method. 293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)Cell surface staining and flow cytometry analysis: 293T cells transfected with a Spike expressor or 293T-Spike cells were stained with the anti-RBD CR3022 monoclonal Ab (5 μg/ml) or plasma (1:250 dilution). 293T-Spikesuggested: NoneSoftware and Algorithms Sentences Resources Samples were acquired on a LSRII cytometer (BD Biosciences) and data analysis was performed using FlowJo v10.5.3 (Tree Star). FlowJosuggested: (FlowJo, RRID:SCR_008520)Statistical analyses: Statistics were analyzed using GraphPad Prism version 8.4.3 (GraphPad, San Diego, CA). GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)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: We found the following clinical trial numbers in your paper:
Identifier Status Title NCT04412486 Recruiting COVID-19 Convalescent Plasma (CCP) Transfusion NCT04342182 Active, not recruiting Convalescent Plasma as Therapy for Covid-19 Severe SARS-CoV-… 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.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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