SARS-CoV-2 hijacks neutralizing dimeric IgA for enhanced nasal infection and injury

Article activity feed

1. 

   ScreenIT

   Oct 9, 2021

   SciScore for 10.1101/2021.10.05.463282: (What is this?)

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

   Table 1: Rigor

   Ethics	Consent: Written informed consent was obtained from all patients. IRB: This study was approved by the Institutional Review Board of The University of Hong Kong/Hospital Authority Hong Kong West Cluster, the Hong Kong East Cluster Research Ethics Committee, and the Kowloon West Cluster Research Ethics Committee (UW 13-265, HKECREC-2018-068, KW/EX-20-038[144-26]).
   Sex as a biological variable	Male and female golden Syrian hamsters (Mesocricetus auratus) (aged 6–10 weeks) were purchased from the Chinese University of Hong Kong Laboratory Animal Service Centre through the HKU Laboratory Animal Unit
   Randomization	The hamsters were randomized from different litters into experimental groups.
   Blinding	not …

   SciScore for 10.1101/2021.10.05.463282: (What is this?)

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

   Table 1: Rigor

   Ethics	Consent: Written informed consent was obtained from all patients. IRB: This study was approved by the Institutional Review Board of The University of Hong Kong/Hospital Authority Hong Kong West Cluster, the Hong Kong East Cluster Research Ethics Committee, and the Kowloon West Cluster Research Ethics Committee (UW 13-265, HKECREC-2018-068, KW/EX-20-038[144-26]).
   Sex as a biological variable	Male and female golden Syrian hamsters (Mesocricetus auratus) (aged 6–10 weeks) were purchased from the Chinese University of Hong Kong Laboratory Animal Service Centre through the HKU Laboratory Animal Unit
   Randomization	The hamsters were randomized from different litters into experimental groups.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Serially diluted plasma from healthy individuals or previously published monoclonal antibodies against HIV-1 (VRC01) were used as negative controls.	HIV-1 suggested: (bNAber Cat# bNAberID_1, RRID:AB_2491019) VRC01 suggested: (bNAber Cat# bNAberID_1, RRID:AB_2491019)
   Two consecutive staining steps were conducted: the first one used an antibody and RBD cocktail incubation of 30 min at 4 °C; the second staining involved staining with anti-His-APC and anti-His-FITC antibodies (Abcam) at 4 °C for 30 min to detect the His tag of the RBD.	anti-His-APC suggested: (Miltenyi Biotec Cat# 130-101-320, RRID:AB_2747411) anti-His-FITC suggested: (Miltenyi Biotec Cat# 130-092-675, RRID:AB_1103226)
   For dIgA antibody production, plasmids of paired heavy chain (IgA1, IgA2) and kappa light chain together with a J chain were co-transfected into Expi293™ expression system (Thermo Fisher Scientific) at the ratio of 1:1:1 following the manufacturer’s instructions.	IgA1 suggested: None IgA2 suggested: None
   Antibodies produced from cell culture supernatants were purified immediately by affinity chromatography using recombinant Protein G-Agarose (Thermo Fisher Scientific) or CaptureSelect™ IgA Affinity Matrix (Thermo Fisher Scientific) according to the manufacturer’s instructions, to purify IgG and IgA, respectively.	IgA suggested: None
   2 mL-fractions were collected, pooled, concentrated and evaluated by western blot using mouse anti-IGJ monoclonal antibody [KT109] (Abcam) and rabbit anti-human IgA alpha chain antibody (Abcam).	anti-IGJ suggested: None anti-human IgA alpha chain antibody ( Abcam) . suggested: None
   The differences in response units between ACE2 injection alone and prior antibody incubation reflect the antibodies’ competitive ability against ACE2 binding to the spike protein.	ACE2 suggested: None
   For identification and localization of SARS-CoV-2 nucleocapsid protein (NP) in organ tissues, immunofluorescence staining was performed on deparaffinized and rehydrated tissue sections using a rabbit anti-SARS-CoV-2-NP protein antibody together with an AF488-conjugated anti-rabbit IgG (Jackson ImmunoResearch, PA, USA).	SARS-CoV-2 nucleocapsid protein (NP suggested: None anti-SARS-CoV-2-NP protein suggested: None anti-rabbit IgG suggested: None
   After blocking with 0.1% Sudan black B for 15 min and 1% bovine serum albumin (BSA)/PBS at RT for 30 min, the primary rabbit anti-SARS-CoV-2-NP antibody (1:4000 dilution with 1% BSA/PBS) was incubated at 4°C overnight.	anti-SARS-CoV-2-NP suggested: None
   For identification of DC-SIGN expression, we stained the NT slices with rabbit anti-DC-SIGN primary antibody (Abcam) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (Life Technologies) according to the manufacturer’s instructions.	anti-DC-SIGN suggested: (IMGENEX Cat# DDX0208A488, RRID:AB_1929964)
   For identification of ACE2 expression, the goat anti-ACE2 primary antibody (R&D) and Alexa Fluor 568 donkey anti-goat IgG (H+L) secondary antibodies (Invitrogen) according to the manufacturer’s instructions.	anti-goat IgG suggested: None
   The infectious medium was replaced with fresh medium containing respective concentration of antibody after washing 3 times with PBS. 24 h later, the infected cells were imaged under fluorescence microscope after staining with AF488-conjugated anti-SARS-CoV-2 NP antibody.	anti-SARS-CoV-2 NP suggested: None
   Cells were further permeabilized with 0.2% Triton X-100 and incubated with cross-reactive rabbit sera anti-SARS-CoV-2-N for 1 hour at RT before adding Alexa Fluor 488 goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (Life Technologies).	anti-SARS-CoV-2-N suggested: None
   After treatment with B8 antibodies at the dose of 3000 ng/ml/mL for 1 hour, HEK293T cells transfected with SARS-CoV-2 spike-GFP were added into the treated Vero-E6 TMPRSS2 cells and co-cultured for 48 hours.	B8 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   The viral challenge experiments were then conducted in our Biosafety Level-3 animal facility following SOPs strictly, with strict adherence to SOPs Cell lines: HEK293T cells, HEK293T-hACE2 cells Vero-E6 cells, HK2 cells and Vero-E6-TMPRSS2 cells were maintained in DMEM containing 10% FBS, 2 mM L-glutamine, 100 U/mL/mL penicillin and incubated at 37 □ in a 5% CO2 setting 62	HEK293T-hACE2 suggested: RRID:CVCL_A7UK) Vero-E6 suggested: None Vero-E6-TMPRSS2 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Briefly, The pseudovirus was generated by co-transfection of 293T cells with pVax-1-S-COVID19 and pNL4-3Luc_Env_Vpr, carrying the optimized spike (S) gene (QHR63250) and a human immunodeficiency virus type 1 backbone, respectively 77	293T suggested: None
   The antibody-virus mixtures were subsequently added to pre-seeded HEK 293T-ACE2 cells.	HEK 293T-ACE2 suggested: RRID:CVCL_A7UK)
   Mixtures were then transferred to 96-well plates pre-seeded with 1×104/well Vero E6 cells and incubated at 37°C for 24 hours.	Vero E6 suggested: None
   The HEK293T-CD209 cells were pre-treated with 10 ng/ml/mL of B8-dIgA or control dIgA and incubated for 6 h prior SARS-CoV-2 infection (MOI=0.05).	HEK293T-CD209 suggested: None
   Effects of B8 antibodies on SARS-CoV-2 mediated cell-cell fusion: Vero-E6 TMPRSS2 cells were seeded into 48-well plates and cultured overnight.	Vero-E6 TMPRSS2 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   After treatment with B8 antibodies at the dose of 3000 ng/ml/mL for 1 hour, HEK293T cells transfected with SARS-CoV-2 spike-GFP were added into the treated Vero-E6 TMPRSS2 cells and co-cultured for 48 hours.	HEK293T suggested: None
   Recombinant DNA
   Sentences	Resources
   Briefly, The pseudovirus was generated by co-transfection of 293T cells with pVax-1-S-COVID19 and pNL4-3Luc_Env_Vpr, carrying the optimized spike (S) gene (QHR63250) and a human immunodeficiency virus type 1 backbone, respectively 77	pVax-1-S-COVID19 suggested: None pNL4-3Luc_Env_Vpr suggested: None
   Software and Algorithms
   Sentences	Resources
   Sequences were aligned using Clustal W in the BioEdit sequence analysis package (Version 7.2)	BioEdit suggested: (BioEdit, RRID:SCR_007361)
   Half-maximal (IC50) or 90% (IC90) inhibitory concentrations of the evaluated antibody were determined by inhibitor vs. normalized response -- 4 Variable slope using GraphPad Prism 6 or later (GraphPad Software Inc.)	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   The defocus value of each image, which was set from −1.0 to −2.0 μm during data collection, was determined by Gctf.	Gctf suggested: (GCTF, RRID:SCR_016500)
   Sequential data processing was carried out on RELION 3.0 and RELION 3.1.	RELION suggested: (RELION, RRID:SCR_016274)
   RBD-Fab maps were fitted onto the whole structure map using Chimera, then combined using PHENIX combine_focused_maps.	PHENIX suggested: (Phenix, RRID:SCR_014224)
   Model building and structure refinement: The spike model (PDB code: 6VSB) and the initial model of the B8 Fab generated by SWISS-Model were fitted into the EM density map, and further manually adjusted with Coot.	Coot suggested: (Coot, RRID:SCR_014222)
   The final structures were validated using Phenix.molprobity	molprobity suggested: (MolProbity, RRID:SCR_014226)
   . UCSF Chimera, ChimeraX and PyMol were used for map segmentation and figure generation.	PyMol suggested: (PyMOL, RRID:SCR_000305)
   NP+ cells per field were quantified based on the mean fluorescence intensity (MFI) using the ZEN BLACK 3.0 and ImageJ (NIH).	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   The fluorescence density of SARS-CoV-2 infected cells was acquired using a Sapphire Biomolecular Imager (Azure Biosystems) and then the MFI of four randomly selected areas of each sample was quantified using Fiji software (NIH).	Fiji suggested: (Fiji, RRID:SCR_002285)
   Re-analysis of published nasal brushing single-cell data: The preprocessed scRNA-seq data from nasal brushing samples of 2 healthy controls and 4 COVID-19 patients were downloaded from Gene Expression Omnibus (GEO) database with accession numbers GSE171488 and GSE164547.	Gene Expression Omnibus suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)
   Quantification and statistical analysis: Statistical analysis was performed using PRISM 6.0 or later.	PRISM suggested: (PRISM, RRID:SCR_005375)

   ---

   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: We detected the following sentences addressing limitations in the study:

   One limitation is the insufficient amounts of HuNAbs distributed on the nasal mucosal surface for protection 47. Other reasons might include alternative entry pathways engaged by SARS-CoV-2 to evade HuNAbs. To this end, Liu et al. reported recently that antibodies against the spike N-terminal domain (NTD) induced an open conformation of the RBD and thus enhanced the binding capacity of the spike to the ACE2 receptor, leading to increased viral infectivity 66. Yeung et al. demonstrated nicely that SARS-CoV-2 could engage soluble ACE2 (sACE2) and then bind alternate receptors for viral entry, through interaction between a spike/sACE2 complex with the angiotensin II AT1 receptor, or interaction between a spike/sACE2/vasopressin complex with the AVPR1B vasopressin receptor, respectively 49. In this study, we found that, in the presence of potent neutralizing B8-dIgA1 or B8-dIgA2 antibodies, SARS-CoV-2 used the cellular receptor CD209 for capture or infection, which likely expanded the use of CD209+ cells as target cells, leading to enhanced NT infection and trans-infection. Interestingly, a preprint report suggests that cells expressing CD209 can be infected directly by SARS-CoV-2 through an interaction of the spike with the NTD instead of the RBD 67. This mode of action, however, was unlikely to explain our findings, because no enhancement of SARS-CoV-2 nasal infection was found in presence of control dIgA1 and dIgA2. Our results rather suggest that the direct binding of virus-b...

   ---

   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.

   ---

   Read the original source
2. 

   Version published to 10.1101/2021.10.05.463282v1 on bioRxiv

   Oct 6, 2021