A recombinant fragment of Human surfactant protein D binds Spike protein and inhibits infectivity and replication of SARS-CoV-2 in clinical samples
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Abstract
Rationale
COVID-19 is an acute infectious disease caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Human surfactant protein D (SP-D) is known to interact with spike protein of SARS-CoV, but its immune-surveillance against SARS-CoV-2 is not known.
Objective
This study aimed to examine the potential of a recombinant fragment of human SP-D (rfhSP-D) as an inhibitor of replication and infection of SARS-CoV-2.
Methods
rfhSP-D interaction with spike protein of SARS-CoV-2 and hACE-2 receptor was predicted via docking analysis. The inhibition of interaction between spike protein and ACE-2 by rfhSP-D was confirmed using direct and indirect ELISA. The effect of rfhSP-D on replication and infectivity of SARS-CoV-2 from clinical samples was studied by measuring the expression of RdRp gene of the virus using qPCR.
Measurements and Main Results
In-silico interaction studies indicated that three amino acid residues in the RBD of spike of SARS-CoV-2 were commonly involved in interacting with rfhSP-D and ACE-2. Studies using clinical samples of SARS-CoV-2 positive cases (asymptomatic, n=7 and symptomatic, n=8 and negative controls n=15) demonstrated that treatment with 5μM rfhSP-D inhibited viral replication by ~5.5 fold and was more efficient than Remdesivir (100 μM). Approximately, a 2-fold reduction in viral infectivity was also observed after treatment with 5μM rfhSP-D.
Conclusions
These results conclusively demonstrate that the calcium independent rfhSP-D mediated inhibition of binding between the receptor binding domain of the S1 subunit of the SARS-CoV-2 spike protein and human ACE-2, its host cell receptor, and a significant reduction in SARS-CoV-2 infection and replication in-vitro .
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SciScore for 10.1101/2020.12.18.423415: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding The therapeutic agent, rfhSP-D trimer, (PDB id: 1PW9) was individually blind docked with the structure of RBD of S protein in the open conformation (PDB id: 6VYB) and dimeric ACE-2 (PDB id: 6VW1) using Patchdock. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources , anti-rabbit IgG HRP (Cat # 31466, Invitrogen) or Protein A HRP (Cat # 18-160, Merck) at 1: 5000 dilution was used secondary antibodies by adding them to the respective wells of the appropriate primary antibodies and incubating them for 1 h at 37°C. anti-rabbit…SciScore for 10.1101/2020.12.18.423415: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding The therapeutic agent, rfhSP-D trimer, (PDB id: 1PW9) was individually blind docked with the structure of RBD of S protein in the open conformation (PDB id: 6VYB) and dimeric ACE-2 (PDB id: 6VW1) using Patchdock. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources , anti-rabbit IgG HRP (Cat # 31466, Invitrogen) or Protein A HRP (Cat # 18-160, Merck) at 1: 5000 dilution was used secondary antibodies by adding them to the respective wells of the appropriate primary antibodies and incubating them for 1 h at 37°C. anti-rabbit IgGsuggested: (Thermo Fisher Scientific Cat# 31466, RRID:AB_10960844)A similar experiment was carried out in parallel using rfhSP-D (20, 10 and 5 μg/ml), supplemented with 10mM EDTA and probed with polyclonal antibodies against SPD (1:5000) to evaluate if the S protein-rfhSP-D binding was calcium-dependent. SPDsuggested: NoneExperimental Models: Cell Lines Sentences Resources Vero Cell Infection Assay: Vero cell line (derived from African green monkey epithelial Kidney cells) (ATCC® CCL–81™) (5×104) were cultured for 16 h in each well of a 12 well plate in serum-free medium (MEM Glutamax, containing 1% v/v Penicillin-Streptomycin and 1%v/v sodium pyruvate [Gibco, Thermofisher]). Verosuggested: RRID:CVCL_ZW93)Software and Algorithms Sentences Resources The receptor and ligand were then re-docked using Patchdock web server (14, 15) to validate the docking protocol. Patchdocksuggested: (PatchDock, RRID:SCR_017589)Statistical Analysis: Graphs were generated using GraphPad Prism 8.0 software, and the statistical analysis was performed using a two-way ANOVA test. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: Thank you for sharing your data.
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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
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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