Anti-chemokine antibodies after SARS-CoV-2 infection correlate with favorable disease course
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Abstract
Infection by SARS-CoV-2 leads to diverse symptoms, which can persist for months. While antiviral antibodies are protective, those targeting interferons and other immune factors are associated with adverse COVID-19 outcomes. Instead, we discovered that antibodies against specific chemokines are omnipresent after COVID-19, associated with favorable disease, and predictive of lack of long COVID symptoms at one year post infection. Anti-chemokine antibodies are present also in HIV-1 infection and autoimmune disorders, but they target different chemokines than those in COVID-19. Monoclonal antibodies derived from COVID- 19 convalescents that bind to the chemokine N-loop impair cell migration. Given the role of chemokines in orchestrating immune cell trafficking, naturally arising anti-chemokine antibodies associated with favorable COVID-19 may be beneficial by modulating the inflammatory response and thus bear therapeutic potential.
One-Sentence Summary:
Naturally arising anti-chemokine antibodies associate with favorable COVID-19 and predict lack of long COVID.
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SciScore for 10.1101/2022.05.23.493121: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: The study was performed in compliance with all relevant ethical regulations and the study protocols were approved by the Ethical Committee of the Canton Ticino (ECCT): CE-3428 and CE-3960.
Consent: Written informed consent was obtained from all participants, and all samples were coded to remove identifiers at the time of blood withdrawal.Sex as a biological variable not detected. Randomization All assays were done in triplicate, and for each well the migrated cells were counted at 100-fold magnification in 5 randomly selected high-power fields (5HPF). Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Chemotaxis … SciScore for 10.1101/2022.05.23.493121: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: The study was performed in compliance with all relevant ethical regulations and the study protocols were approved by the Ethical Committee of the Canton Ticino (ECCT): CE-3428 and CE-3960.
Consent: Written informed consent was obtained from all participants, and all samples were coded to remove identifiers at the time of blood withdrawal.Sex as a biological variable not detected. Randomization All assays were done in triplicate, and for each well the migrated cells were counted at 100-fold magnification in 5 randomly selected high-power fields (5HPF). Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Chemotaxis was performed with preB 300.19 expressing CCR2, at a final IgG concentration of 200 µg/ml, in the presence of the chemokine concentration resulting in peak migration when no antibodies were added (CCL2 [10nM], CCL7 [100nM], CCL8 [100nM]). CCR2suggested: NoneCCL2suggested: NoneCCL7suggested: NoneCCL8suggested: NonePlates were subsequently washed 4 times with washing buffer and incubated with anti-human IgG secondary antibody conjugated to horseradish peroxidase (HRP) (GE Healthcare, NA933) at a 1:5000 dilution in PBS + 0.05% Tween-20. anti-human IgGsuggested: (GE Healthcare Cat# NA933-1ml, RRID:AB_772208)Kinetic of signature anti-chemokine IgG antibodies (fig. anti-chemokine IgGsuggested: NoneSingle cell sorting by flow cytometry: B cells were enriched from PBMCs of uninfected controls or of COVID-19 convalescent individuals 6 months after COVID-19 (participant CLM9 for anti-CCL8 antibodies; CLM64 for anti-CCL20 antibodies; CLM5, CLM7 and CLM33 for anti-CXCL13 antibodies; and CLM8 and CLM30 for anti-CXCL16 antibodies), using the pan-B-cell isolation kit according to manufacturer’s instructions (Miltenyi Biotec, 130-101-638). anti-CCL20suggested: Noneanti-CXCL13suggested: Noneanti-CXCL16suggested: NoneThe enriched B cells were subsequently stained in FACS buffer (PBS + 2% FCS + 1mM EDTA) with the following antibodies/reagents (all 1:200 diluted) for 30 min on ice: anti-CD20-PE-Cy7 (BD Biosciences, 335828), anti-CD14-APC-eFluor 780 (Thermo Fischer Scientific, 47-0149-42), anti-CD16-APC-eFluor 780 (Thermo Fischer Scientific, 47-0168-41), anti-CD3-APC-eFluor 780 (Thermo Fischer Scientific, 47-0037-41), anti-CD8-APC-eFluor 780 (Invitrogen, 47-0086-42) anti-CD20-PE-Cy7suggested: Noneanti-CD14-APC-eFluor 780suggested: Noneanti-CD16-APC-eFluor 780suggested: Noneanti-CD3-APC-eFluor 780suggested: Noneanti-CD8-APC-eFluor 780suggested: NoneAmplicons from this first PCR reaction served as templates for sequence and ligation independent cloning (SLIC) into human IgG1 antibody expression vectors. human IgG1suggested: NoneWhere indicated, the anti-Zika virus monoclonal antibody Z021 (75) was used as an isotype control. anti-Zikasuggested: NoneExperimental Models: Cell Lines Sentences Resources SARS-CoV-2 pseudotyped reporter virus and neutralization assay: To generate (HIV-1/NanoLuc2AEGFP)-SARS-CoV-2 particles, HEK293T cells were co- transfected with the three plasmids pHIVNLGagPol, pCCNanoLuc2AEGFP, and SARS- CoV- 2 S as described elsewhere (47, 80) HEK293Tsuggested: NoneBriefly, threefold serially diluted plasma samples (from 1:50 to 1:328’050) were incubated with SARS-CoV- 2 pseudotyped virus for 1h at 37 °C, and the virus-plasma mixture was subsequently incubated with 293TACE2 cells for 48 h. 293TACE2suggested: NoneRecombinant DNA Sentences Resources SARS-CoV-2 pseudotyped reporter virus and neutralization assay: To generate (HIV-1/NanoLuc2AEGFP)-SARS-CoV-2 particles, HEK293T cells were co- transfected with the three plasmids pHIVNLGagPol, pCCNanoLuc2AEGFP, and SARS- CoV- 2 S as described elsewhere (47, 80) pHIVNLGagPolsuggested: NonepCCNanoLuc2AEGFPsuggested: NoneSoftware and Algorithms Sentences Resources S5E): Experiments were performed with plasma samples assayed at a 1:50 dilution side-by-side on the same plate, and the average optical density at 450 nm obtained from two independent experiments was plotted with GraphPad Prism. GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Live single Zombie-NIR−CD14−CD16−CD3−CD8−CD20+Ova−N-loop-PE+N-loop- AF647+ B cells were single-cell sorted into 96-well plates containing 4 μl of lysis buffer (0.5× PBS, 10 mM DTT, 3,000 units/ml RNasin Ribonuclease Inhibitors [Promega, N2615]) per well using a FACS Aria III, and the analysis was performed with FlowJo software. FlowJosuggested: (FlowJo, RRID:SCR_008520)1 was generated from the structure of inactive CCR2 (PDB code: 5T1A) (81), together with the electron microscopy structures of CCR5 and CCR6 (PDB codes: 6MEO and 6WWZ, respectively (82, 83) by using SWISS-MODEL (84) server and the molecular graphics program PyMOL 2.5.0 for modeling the N- and C-terminus of the receptor. PyMOLsuggested: (PyMOL, RRID:SCR_000305)t-SNE: t-SNE analysis was performed using the Rtsne R package v 0.15 (https://CRAN.R-project.org/package=Rtsne) using the AUC values for all chemokines. Rtsnesuggested: (Rtsne, RRID:SCR_016342)Results from OddPub: Thank you for sharing your code.
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.
- 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.
Results from scite Reference Check: We found no unreliable references.
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