Longitudinal characterisation of phagocytic and neutralisation functions of anti-Spike antibodies in plasma of patients after SARS-CoV-2 infection

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   ScreenIT

   Dec 24, 2021

   SciScore for 10.1101/2021.12.21.473774: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	IRB: The study protocol was approved by the Human Research Ethics Committees of the Northern Sydney Local Health District, the University of New South Wales, NSW Australia (ETH00520), CALHN Human Research Ethics Committee, Adelaide, Australia Consent: Written informed consent was obtained from all participants before enrolment.
   Sex as a biological variable	Archival serum or plasma collected from 25 healthy donors prior to the pandemic with an age range of 24-73 years, and a male to female ratio of 1:2.4 was used as controls.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Expression of …

   SciScore for 10.1101/2021.12.21.473774: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	IRB: The study protocol was approved by the Human Research Ethics Committees of the Northern Sydney Local Health District, the University of New South Wales, NSW Australia (ETH00520), CALHN Human Research Ethics Committee, Adelaide, Australia Consent: Written informed consent was obtained from all participants before enrolment.
   Sex as a biological variable	Archival serum or plasma collected from 25 healthy donors prior to the pandemic with an age range of 24-73 years, and a male to female ratio of 1:2.4 was used as controls.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Expression of surface Fc-receptors was assessed by flow cytometry using monoclonal antibodies (mAbs) against Fcγ receptor RI (CD64)-FITC, FcγRIII (CD16)-PE, CD14-PerCP	CD64)-FITC suggested: None
   Endpoint titre and Isotyping of anti-SARS-CoV-2 Spike and anti-RBD antibodies in sera of patients with SARS-CoV-2 infection: Anti-SARS-CoV-2 Spike and RBD IgG antibody in sera was quantified using a modified direct ELISA [27].	anti-SARS-CoV-2 suggested: None RBD IgG suggested: None
   For the endpoint titre of total anti-Spike or anti-RBD antibodies, 50μL of horseradish peroxidase (HRP)-conjugated mouse anti-human detection antibody added per well (Jackson ImmunoResearch, USA) (1:5000 dilution in 5% skim milk).	anti-Spike suggested: None anti-RBD suggested: None anti-human detection suggested: None
   For isotyping of the anti-Spike or anti-RBD antibodies, 50μL/well of HRP-conjugated immunoglobulin subtype or IgG subclass specific detection antibodies were added.	IgG subclass specific detection antibodies suggested: None
   These include 1:6000 dilution of anti-total human IgG (Jackson Immunoresearch), 1:3000 dilution of anti-human IgA (α-chain-specific) (Sigma) or 1:3000 dilution of anti-human IgM (μ-chain-specific) (Sigma) antibody subtypes and 1:6000 dilution of anti-human IgG1, IgG2, IgG3 or IgG4 (Southern Biotech, USA).	anti-total human IgG suggested: (Cell Signaling Technology Cat# 13123, RRID:AB_2617178) anti-human IgA ( α-chain-specific ) ( Sigma ) suggested: None anti-human IgM ( μ-chain-specific ) ( Sigma ) suggested: None anti-human IgG1 suggested: None IgG2 suggested: None IgG3 suggested: None IgG4 suggested: None
   To assess whether the uptake of the microbeads opsonised with the plasma of patients with acute disease was via Fc-receptor (antibody-dependent), and other heat labile opsonins such as complements (eg C3b), either the Fc-receptors on the THP-1 cells were pre-blocked using the universal Fc receptor blocking agent (Miltenyi, USA) [23], or the plasma heat inactivated at 56°C for 30 minutes, as described [28].	antibody-dependent) , suggested: None
   Surface plasmon resonance: Surface plasmon resonance (SPR) was performed using a Biacore T200 (Cytiva, USA) to determine the binding characteristics of the anti-Spike polyclonal antibodies in patient plasma to SARS-CoV-2 Spike antigen.	SARS-CoV-2 Spike antigen . suggested: None
   Friedman’s test with pairwise Dunn’s tests were used to compare the repeated measures of Spike p-score, Spike and RBD end point titre, neutralisation titre and antibody affinity across the timepoints V1, V2 and V3.	V3 suggested: None
   To rank the variables of statistical importance that associated with Spike p-score (dependent variable), multiple linear regression analysis was performed using disease severity, age, gender, DPS, anti-Spike/RBD endpoint antibody titre, anti-Spike/RBD antibody subtypes and neutralisation titre as independent variables.	anti-Spike/RBD suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   To assess whether the uptake of the microbeads opsonised with the plasma of patients with acute disease was via Fc-receptor (antibody-dependent), and other heat labile opsonins such as complements (eg C3b), either the Fc-receptors on the THP-1 cells were pre-blocked using the universal Fc receptor blocking agent (Miltenyi, USA) [23], or the plasma heat inactivated at 56°C for 30 minutes, as described [28].	THP-1 suggested: None
   Retroviral SARS-CoV-2 Spike pseudovirus were generated in 293T cells by co-transfecting expression plasmids containing SARS-CoV-2 Spike and MLV gag/pol and luciferase vectors using Calphos transfection kit (Takara Bio, USA) as described [20].	293T suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
   Briefly, pseudovirus were incubated for one hour with heat inactivated (56 °C for 30 minutes) patient serum prior to infecting 293T-ACE2 cells (kindly provided by A/Prof Jesse Bloom) by a two hour spinoculated at 800xg in 96-well white flat bottom plates in triplicates (Sigma-Aldrich, USA)	293T-ACE2 suggested: None
   Recombinant DNA
   Sentences	Resources
   Biotinylated recombinant SARS-CoV-2 Spike and RBD proteins: SARS-CoV-2 Wuhan-Hu-1 (GenPept: QJE37812) RBD protein (amino acid residues 319–541) and Spike protein (amino acid residue 15-1213) were cloned into pCEP4 mammalian expression vector containing N-terminal human Ig kappa leader sequence and C-terminal Avi-tag and His-tag (Invitrogen, USA)	pCEP4 suggested: RRID:Addgene_16479)
   Software and Algorithms
   Sentences	Resources
   After a two hours incubation, cells were washed once with 1mL of cold PBS containing 0.5% FBS and 0.005% of sodium azide and gentle centrifugation at 335xg for five minutes at 4°C, fixed in 400μL of 1% paraformaldehyde and kept at 4°C in the dark until acquisition of data using BD FACSCalibur™ Flow cytometer.	BD FACSCalibur™ suggested: (BD FACSCalibur Flow Cytometry System, RRID:SCR_000401)
   A total of 2×104 events were acquired and the proportions of cells that phagocytosed the beads (% of cells that took up the beads) and their fluorescent intensities (amounts of beads taken up per cell) were analysed using BD FlowJo version 10.5.0 software.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   All the images were Airyscan processed with Zen Black Edition (Zeiss Software).	Zen suggested: None
   Relative Luminescence Unit (RLU) in cell lysates was measured using CLARIOstar microplate reader (BMG Labtech, Australia), percentage neutralisation of Spike pseudovirus determined and the fifty percent inhibitory (ID50) calculated using non-linear regression model (GraphPad Prism version 9.0) [29]	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   Kinetic constants, including association constant (Ka), equilibrium constant (KD) (affinity) and dissociation constant (Ka) (avidity), were calculated using BIAcore evaluation software version 4.1 [30].	BIAcore suggested: (Biacore T100 System, RRID:SCR_019679)
   Statistical analysis: All data were analysed with Prism Software (version 9.0, GraphPad, USA)	Prism suggested: (PRISM, RRID:SCR_005375)

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   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).

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   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.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   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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   Results from scite Reference Check: We found no unreliable references.

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   Version published to 10.1101/2021.12.21.473774v1 on bioRxiv

   Dec 22, 2021