Elucidating Design Principles for Engineering Cell‐Derived Vesicles to Inhibit SARS‐CoV‐2 Infection

Abstract

The ability of pathogens to develop drug resistance is a global health challenge. Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) presents an urgent need wherein several variants of concern resist neutralization by monoclonal antibody (mAb) therapies and vaccine‐induced sera. Decoy nanoparticles—cell‐mimicking particles that bind and inhibit virions—are an emerging class of therapeutics that may overcome such drug resistance challenges. To date, quantitative understanding as to how design features impact performance of these therapeutics is lacking. To address this gap, this study presents a systematic, comparative evaluation of various biologically derived nanoscale vesicles, which may be particularly well suited to sustained or repeated administration in the clinic due to low toxicity, and investigates their potential to inhibit multiple classes of model SARS‐CoV‐2 virions. A key finding is that such particles exhibit potent antiviral efficacy across multiple manufacturing methods, vesicle subclasses, and virus‐decoy binding affinities. In addition, these cell‐mimicking vesicles effectively inhibit model SARS‐CoV‐2 variants that evade mAbs and recombinant protein‐based decoy inhibitors. This study provides a foundation of knowledge that may guide the design of decoy nanoparticle inhibitors for SARS‐CoV‐2 and other viral infections.

SciScore for 10.1101/2021.12.04.471153: (What is this?)

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

Table 1: Rigor

Ethics	not detected.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
After decanting the supernatant, cells were resuspended in 50 μL FACS buffer and blocked with 10 μL of 1 mg/mL IgG (Thermo Fisher, Human IgG Isotype Control, 02-7102, RRID: AB_2532958) for 5 min at 4°C.	Human IgG Isotype Control detected: (Thermo Fisher Scientific Cat# 02-7102, RRID:AB_2532958)
After blocking, 2.5 μL of 0.2 μg/μL α-ACE2 antibody from R&D Systems (Human ACE-2 Alexa Fluor® 488-conjugated Antibody, FAB9332G-100UG) was added and incubated for 30 min at 4°C.	α-ACE2 suggested: …

SciScore for 10.1101/2021.12.04.471153: (What is this?)

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

Table 1: Rigor

Ethics	not detected.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
After decanting the supernatant, cells were resuspended in 50 μL FACS buffer and blocked with 10 μL of 1 mg/mL IgG (Thermo Fisher, Human IgG Isotype Control, 02-7102, RRID: AB_2532958) for 5 min at 4°C.	Human IgG Isotype Control detected: (Thermo Fisher Scientific Cat# 02-7102, RRID:AB_2532958)
After blocking, 2.5 μL of 0.2 μg/μL α-ACE2 antibody from R&D Systems (Human ACE-2 Alexa Fluor® 488-conjugated Antibody, FAB9332G-100UG) was added and incubated for 30 min at 4°C.	α-ACE2 suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Cell lines and cell culture: HEK293FT cells were purchased from Thermo Fisher/Life Technologies.	HEK293FT suggested: RRID:CVCL_6911)
Pseudotype virus production: HEK293FT or Lenti-X HEK293T cells (Lenti-X) were used to produce SARS-CoV-2 pseudotyped lentivirus (Spike-lenti) for optimizing viral production; Lenti-X cells were used to generate Spike-lenti for all viral inhibition experiments.	HEK293T suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
Recombinant DNA
Sentences	Resources
The Spike protein from pcDNA3.1-SARS2-Spike was a gift from Fang Li (Addgene plasmid # 145032; http://n2t.net/addgene:145032; RRID:Addgene_145032);34 this gene was cloned into a modified pcDNA 3.1 backbone (Clontech-Takara) with a beta-globin intron in the 5’ untranslated region for pseudotyping lentivirus.	detected: RRID:Addgene_145032) pcDNA 3.1 suggested: RRID:Addgene_20407)
The Tet3G transactivator (pLVX-EF1a-TET3G) and cognate TRE3GV promoter (pLVX-TRE3G	pLVX-EF1a-TET3G suggested: None
) (Takara) were cloned into modified pGIPZ and pLVX (Takara) backbones, respectively.	pGIPZ suggested: RRID:Addgene_121488) pLVX suggested: RRID:Addgene_174088)
Cells were then transfected via calcium phosphate method.51 Briefly, DNA (3 μg pMD2.	pMD2 suggested: None
G encoding vesicular stomatitis virus G protein (VSV-G), 8 μg psPAX2 packaging vector, and 10 μg of transfer plasmid encoding desired transgene) were diluted with sterile H2O and added to 2M CaCl2 to achieve a final concentration of 0.3M CaCl2.	psPAX2 suggested: RRID:Addgene_12260)
Software and Algorithms
Sentences	Resources
Cell lines and cell culture: HEK293FT cells were purchased from Thermo Fisher/Life Technologies.	Thermo Fisher/Life suggested: None
53 Band intensities from ImageJ for the sACE2 standards were analzyed in MATLAB (Mathworks, R2021b) as a function of the amount of ACE2 added in number of molecules (assuming a 115 KDa size for sACE2), and a linear regression was performed to generate a calibration curve.	ImageJ suggested: (ImageJ, RRID:SCR_003070) MATLAB suggested: (MATLAB, RRID:SCR_001622)
Approximately 5,000-10,000 single cells were analyzed for each sample on FlowJo software v10.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
In dose-response curve experiments, the percent of transduced cells for a given treatment was normalized by the percent of transduced cells determined from that treatment’s largest dilution as depicted in Figure S5.54 Curves were then fit with a four parameter, nonlinear regression in GraphPad Prism 9.2.	GraphPad Prism suggested: (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: 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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Elucidating Design Principles for Engineering Cell‐Derived Vesicles to Inhibit SARS‐CoV‐2 Infection

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