Journal: Cellular and Molecular Life Sciences

Article Title: Snake venom phospholipase A 2 s exhibit strong virucidal activity against SARS-CoV-2 and inhibit the viral spike glycoprotein interaction with ACE2

doi: 10.1007/s00018-021-03985-6

Figure Lengend Snippet: Molecular models for HDP-2P (gold) complexes with ACE2 (red), and RBD (turquoise) obtained by protein docking. A Structures of HDP-2P complexed with ACE2 (left panel) and S-protein RBD (right panel) determined using Frodock. The central (control) panel shows re-docking of ACE2 to RBD, which results in a structure that resembles published X-ray structures; B A Rosetta-optimized binding interface between HDP-2P and RBD intersects the ACE2-RBD binding surface, which could prevent RBD from interacting with ACE2; C A different Rosetta-optimized binding interface between HDP-2P and ACE2 where HDP-2P docks away from the ACE2-RBD binding surface, implying no direct interplay between viral protein and PLA 2

Article Snippet: Recombinant human ACE2 (Cusabio, Houston, TX, USA) was immobilized on 3D carboxymethyl dextran hydrogel-coated sensor slides.

Techniques: Control, Binding Assay

Journal: Cellular and Molecular Life Sciences

Article Title: Snake venom phospholipase A 2 s exhibit strong virucidal activity against SARS-CoV-2 and inhibit the viral spike glycoprotein interaction with ACE2

doi: 10.1007/s00018-021-03985-6

Figure Lengend Snippet: PLA 2 s exhibit antiviral activity against viruses containing mutations and interfere with different stages of the SARS-CoV-2 replication cycle. a CPE inhibition assay. Vero E6 cells were infected with SARS-CoV-2 strain PMVL-19 or PMVL-20 at 100 TCID 50 in the presence of different concentrations of HDP-2 for 72 h. CPE inhibition was then measured by colorimetric assay with MTT. b Time-of-drug-addition assay. Vero E6 cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01, and virus yield in the infected cell supernatants was quantified by qRT-PCR 18 h after infection. c 293T/ACE2 were infected with different pseudo-SARS-CoV-2-GFP either in the presence of vehicle (PBS, Control) or HDP-2 (10 µg/ml). Representative fluorescent microscopy images of 293T/ACE2 cells infected with the pseudo-SARS-CoV-2 and treated with HDP-2. HDP-2 treatment led to a decrease in the entry of pseudoviruses, which was manifested in a decrease in the number of GFP-positive cells compared to the control. Scale bars, 100 µm. d Infectivity of pseudo-SARS-CoV-2 particles on 293T/ACE2 cells was quantified by measuring GFP fluorescence. Wuhan, B.1.1.7 and B.1.351 are the Wuhan reference strain, the lineage B.1.1.7 (United Kingdom) and the lineage B.1.351 (South Africa), respectively. Significant difference was determined using a Student’s t test: *p ˂0.05; **p ˂0.01; ***p ˂0.001. All results are shown as mean ± SD of n = 3 or 5 biologically independent samples. RFU relative fluorescence units

Article Snippet: Recombinant human ACE2 (Cusabio, Houston, TX, USA) was immobilized on 3D carboxymethyl dextran hydrogel-coated sensor slides.

Techniques: Activity Assay, Inhibition, Infection, Colorimetric Assay, Virus, Quantitative RT-PCR, Control, Microscopy, Fluorescence

Journal: Cellular and Molecular Life Sciences

Article Title: Snake venom phospholipase A 2 s exhibit strong virucidal activity against SARS-CoV-2 and inhibit the viral spike glycoprotein interaction with ACE2

doi: 10.1007/s00018-021-03985-6

Figure Lengend Snippet: HDP-2P reduces the binding of an anti-ACE2 antibody and RBD of glycoprotein S to ACE2 receptor at 293T/ACE2 cells. a , b Inhibition of anti-ACE2 antibody binding to 293T/ACE2 cells by HDP-2P. 293T/ACE2 cells were incubated with (100 µg/ml) or without (control) HDP-2P for 30 min and stained using human phycoerythrin (PE) conjugated anti-mouse ACE2 antibody. MFI mean fluorescence intensity. c , d Cells were incubated with PBS (control) or HDP-2P. The RBD protein fused with human Fc was then added for 1 h. After washing, the binding of RBD was detected using a DyLight 650-conjugated secondary anti-human Fc antibody. e Representative RBD binding profile in control and HDP-2P-treated cells. The cell populations binding high (RBD hi ) and low (RBD lo ) amount of RBD are shown. The percentage of positive cells was determined using flow cytometry analysis. Significant difference was determined using a Student’s t test: *p ˂0.05, ** p ˂0.01. Results are mean ± SD and are representative of at least three independent determinations

Article Snippet: Recombinant human ACE2 (Cusabio, Houston, TX, USA) was immobilized on 3D carboxymethyl dextran hydrogel-coated sensor slides.

Techniques: Binding Assay, Inhibition, Incubation, Control, Staining, Fluorescence, Flow Cytometry

Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: Summary of sero-negative, acute, and convalescent-phase of COVID-19 patients from which the plasma ACE2 + EVs and RBD-IgG levels were measured.

Article Snippet: The parent ACE2 − human embryonic kidney HEK-293 cells (HEK) (ATCC, CRL-1573) or human cervical cancer HeLa cells (HeLa) (ATCC, CRM-CCL-2) are transduced with a lentiviral pDual-ACE2 expression vector for stable ACE2 expression and production of ACE2 + EVs.

Techniques: Clinical Proteomics, Sampling

Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: a ACE2+ EVs detected in human plasma samples of sero-negative controls (light blue), acute phase (dark green), and convalescent COVID-19 patients (green). One-tail t test (* p = 0.038, ** p = 0.0061 and ** p = 0.0016). Data are presented as mean values ± SEM. b Representative microflow vesiclometry (MFV) plots with gated ACE2+ EVs from sero-negative, acute phase and convalescent COVID-19 patients. c MFV detection of circulating ACE2 + EVs with CD63 + EVs in human plasma of convalescent COVID-19 patient samples (CSB-029 and CSB-023) (green line). Blue line is isotype IgG-negative control. d Flow profiles of ACE2 expression in HEK and HeLa parental control cells (Con, light blue line, ACE2 − ) and with ACE2 overexpression (ACE2, green line). e NanoSight NTA analysis of the sizes of HEK-derived ACE2 − (ev1Con) and ACE2 + (ev1ACE2) and HeLa-derived ACE2 − (ev2Con) and ACE2 + (ev2ACE2). f Immunoblots of HEK and HeLa (ACE2 − and ACE2 + ) EVs and cell lysates for ACE2, TSG101, CD63, CD81, GRP94 and loading control of the membrane proteins upon Ponceau staining. RIPA buffer and Bradford protein assay were used for cells/EVs lysis and protein measurement, respectively ( N = 1 experiment). g Cryo-EM images of HEK-derived EVs, ACE2 − (evCon, left) and ACE2 + (evACE2, right), stained with ACE2 (top) and CD81 (bottom). Scale bars = 100 nm. h Quantified counts of Apogee MFV-based total extracellular vesicles (EVs) and ACE2 + EVs ( N = 2 experiments with n = 6 technical replicates for total EV particles and n = 3 technical replicates for ACE2 + counts). Control EVs are in light blue and ACE2 + EVs in green. Data are presented as mean values +/− SD. i Overlay flow profiles of ACE2 positivity within CD63 + (left column) and CD81+ (right column) EVs isolated from HEK-ACE2 (top row) and HeLa-ACE2 (bottom row) cells, respectively ( n = 3 technical replicates). Light blue line for Control EVs and green line for ACE2 + EVs.

Article Snippet: The parent ACE2 − human embryonic kidney HEK-293 cells (HEK) (ATCC, CRL-1573) or human cervical cancer HeLa cells (HeLa) (ATCC, CRM-CCL-2) are transduced with a lentiviral pDual-ACE2 expression vector for stable ACE2 expression and production of ACE2 + EVs.

Techniques: Clinical Proteomics, Negative Control, Expressing, Control, Over Expression, Derivative Assay, Western Blot, Membrane, Staining, Bradford Protein Assay, Lysis, Cryo-EM Sample Prep, Isolation

Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: a Schematic depiction of the cell-based neutralization assay. b Representative flow profiles showing the percentage (fluorescence mean intensity) of RBD-AF647 binding (at 16 and 3.3 nmol/L) to ACE2 + HEK-293 cells, inhibited by rhACE2 and ACE2 + EVs (evACE2) isolated from HEK-293 and HeLa cells (HEK-EV1 and HeLa-EV2, respectively) whereas ACE2 − EVs (evCon) had no neutralization effects (no RBD in black, PBS in dark blue, rhACE2 in orange, evCon in light blue, and evACE2 in green). c IC 50 of rhACE2 (orange line) and ACE2 in the EVs from ACE2 + HEK (ev1ACE2) and HeLa (ev2ACE2) cells (green lines) on 16 nM RBD-host cell binding (%). GraphPad Prism 9.0.2 was used to calculate the IC 50 . N = 2 experiments with two technical replicates for each. Data are presented as mean values ± SD. d IC 50 of evACE2, ev1 from HEK and ev2 from HeLa cells (green lines), and rhACE2 (orange line) neutralizing infections by wild-type (WT) S + pseudotyped SARS-CoV-2. GraphPad Prism 9.0.2 was used to calculate the IC 50 . N = 2 experiments with two technical replicates for each. Data are presented as mean values ± SD. e IC 50 (nM) of ACE2 in ev1ACE2 (HEK) (green line) and rhACE2 (orange line) upon wild-type SARS-CoV-2 infection. GraphPad Prism 9.0.2 was used to calculate the IC 50 with three biological replicates. Data are presented as mean values ± SD. f Distinct effects of ACE2 + EVs (green lines) and ACE2 − control EVs (light blue line) on inhibiting Vero-6 cell death caused by SARS-CoV-2. N = 2 experiments with three biological replicates each. Data are presented as mean values ± SD. g The IC 50 of ev1ACE2 (HEK) neutralizing infections by pseudotyped SARS-CoV-2 expressing WT (black), B.1.1.7 (α) variant (red), B1.351 (β) variant (dark blue) and B.1.617.2 (δ) (light green) S protein. GraphPad Prism 9.0.2 was used to calculate the IC 50 . N = 2 experiments with two technical replicates each. Data are presented as mean values ± SD. h Effects of ev1ACE2 (HEK) on protecting Vero-6 cell viability against infections of SARS-CoV-2 WT (black), B.1.1.7 (α) variant (red) and B1.351 (β) variant (dark blue) ( n = 3 biological replicates). Data are presented as mean values ± SD.

Article Snippet: The parent ACE2 − human embryonic kidney HEK-293 cells (HEK) (ATCC, CRL-1573) or human cervical cancer HeLa cells (HeLa) (ATCC, CRM-CCL-2) are transduced with a lentiviral pDual-ACE2 expression vector for stable ACE2 expression and production of ACE2 + EVs.

Techniques: Neutralization, Fluorescence, Binding Assay, Isolation, Infection, Control, Expressing, Variant Assay

Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: a Schematic depiction of plasma EV ultracentrifugation and RBD-bead based depletion. b Cryo-EM images of human EV pellets isolated from acute phase COVID-19 plasma (bar = 100 nm). c Immunoblots of plasma EV pellets (sero-negative and COVID-19 acute phase patients CBB-005 and -013) for ACE2 and loading control of protein staining with Ponceau). Laemmli buffer was used for lysis ( N = 1 experiment). d ACE2 + EV pellets from acute phase patients 007, 008, 009, 012, and 013 (CBB) ( n = 2 biological replicates each) blocked SARS-CoV-2 infection-induced death of Vero-6 cells whereas the sero-negative control ( n = 2 biological replicates) and CBB-005 (no detectable ACE2) ( n = 2 biological replicates) did not show neutralization effects. One-tail t test, **** p = 2.24E−08 shown as compared to sero-negative. e , f Levels of ACE2 + EV counts ( n = 3 biological replicates) in plasma EVs (green) and bead-depleted EVs (light blue). One-tail paired t test, * p = 0.011 and ** p = 0.0063 (data are presented as mean values ± SD) ( e ) and altered neutralization effects on RBD–host cell binding ( f ) of the COVID-19 plasma EV pellets prior to and after RBD-bead depletion (convalescent phase CSB-012 and -024; acute phase CBB-008, 009, and 013). One-tail paired t test **** p = 5.11E−05.

Article Snippet: The parent ACE2 − human embryonic kidney HEK-293 cells (HEK) (ATCC, CRL-1573) or human cervical cancer HeLa cells (HeLa) (ATCC, CRM-CCL-2) are transduced with a lentiviral pDual-ACE2 expression vector for stable ACE2 expression and production of ACE2 + EVs.

Techniques: Clinical Proteomics, Cryo-EM Sample Prep, Isolation, Western Blot, Control, Staining, Lysis, Infection, Negative Control, Neutralization, Binding Assay

Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: a Probability of severe disease-free survival in B6.Cg-Tg(K18-ACE2)2Prlmn/J (K18-hACE2) mice receiving SARS-CoV-2 infection (10,000 pfu) and intranasal EVs (130 µg as measured on Nanodrop) per mouse (evCon in light blue and evACE2 in green). Log-rank (Mantel–Cox) and Gehan–Breslow–Wilcoxon tests **** p = 2.27E−07. b Viral loads in mouse lungs on day 5/6 after receiving SARS-CoV-2 infection and administration of evCon ( N = 5 mice) (light blue) or evACE2 ( N = 10 mice) (green). T -test–nonparametric-one tailed, * p = 0.013. Data are presented as mean values ± SD. c . Representative H&E images of mouse lung sections at day 5 or 6 post virus inoculation and EV treatment (evCon and evACE2) intranasally. d , e Acute and chronic inflammation scores ( d ), and alveolar hemorrhage and necrosis scores ( e ) in mouse lungs on day 5/6 after receiving evCon ( N = 5 mice) (light blue) or evACE2 ( N = 7 mice) (green). T -test–nonparametric-one tailed, ** p = 0.005, ** p = 0.003 and *** p = 0.0004. Data are presented as mean values ± SD.

Article Snippet: The parent ACE2 − human embryonic kidney HEK-293 cells (HEK) (ATCC, CRL-1573) or human cervical cancer HeLa cells (HeLa) (ATCC, CRM-CCL-2) are transduced with a lentiviral pDual-ACE2 expression vector for stable ACE2 expression and production of ACE2 + EVs.

Techniques: Infection, One-tailed Test, Virus