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

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the pandemic of the coronavirus induced disease 2019 (COVID-19) with evolving variants of concern. It remains urgent to identify novel approaches against broad strains of SARS-CoV-2, which infect host cells via the entry receptor angiotensin-converting enzyme 2 (ACE2). Herein, we report an increase in circulating extracellular vesicles (EVs) that express ACE2 (evACE2) in plasma of COVID-19 patients, which levels are associated with severe pathogenesis. Importantly, evACE2 isolated from human plasma or cells neutralizes SARS-CoV-2 infection by competing with cellular ACE2. Compared to vesicle-free recombinant human ACE2 (rhACE2), evACE2 shows a 135-fold higher potency in blocking the binding of the viral spike protein RBD, and a 60- to 80-fold higher efficacy in preventing infections by both pseudotyped and authentic SARS-CoV-2. Consistently, evACE2 protects the hACE2 transgenic mice from SARS-CoV-2-induced lung injury and mortality. Furthermore, evACE2 inhibits the infection of SARS-CoV-2 variants (α, β, and δ) with equal or higher potency than for the wildtype strain, supporting a broad-spectrum antiviral mechanism of evACE2 for therapeutic development to block the infection of existing and future coronaviruses that use the ACE2 receptor.

Conflict of interest statement

MD Anderson Cancer Center and R.K. hold patents in the area of exosome biology (unrelated to the topic of this publication) and are licensed to Codiak Biosciences Inc. MD Anderson Cancer Center and R.K. are stock equity holders in Codiak Biosciences Inc. R.K. is a consultant and a scientific advisor of Codiak Biosciences Inc. Northwestern University and H.L., D.F., L.E., A.D.H., and N.K.D. hold issued and/or provisional (on evACE2) patents in the area of exosome therapeutics. H.L., D.F., and A.D.H. are scientific co-founders in ExoMira Medicine Inc. D.B. and J.W. are co-inventors of patents entitled “Active Low Molecular Weight Variants of Angiotensin-Converting Enzyme 2 (ACE2)”, “Active low molecular weight variants of Angiotensin-Converting Enzyme 2 (ACE2) for the treatment of diseases and conditions of the eye” and “Soluble ACE2 Variants and Uses Therefor.” D.B. is the founder of Angiotensin Therapeutics Inc. D.B. has received consulting fees from AstraZeneca, Relypsa, and Tricida, all unrelated to this work. J.W. reports scientific advisor capacity for Angiotensin Therapeutics Inc.

© 2022. The Author(s).

Figures

Fig. 1. Circulating evACE2 increased in the peripheral blood of COVID-19 patients.

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.

Fig. 2. Neutralization effects of evACE2 on RBD-binding and SARS-CoV-2 variant infections.

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 IC50 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 IC50. N = 2 experiments with two technical replicates for each. Data are presented as mean values ± SD. d IC50 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 IC50. N = 2 experiments with two technical replicates for each. Data are presented as mean values ± SD. e IC50 (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 IC50 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 IC50 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 IC50. 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.

Fig. 3. evACE2 in patient plasma neutralizes SARS-CoV-2.

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.

Fig. 4. evACE2 inhibits SARS-COV-2 infection and inflammation in hACE2 transgenic mice.

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.