Engineered extracellular vesicles directed to the spike protein inhibit SARS-CoV-2

Abstract

SARS-CoV-2 (CoV-2) viral infection results in COVID-19 disease, which has caused significant morbidity and mortality worldwide. A vaccine is crucial to curtail the spread of SARS-CoV-2, while therapeutics will be required to treat ongoing and reemerging infections of SARS-CoV-2 and COVID-19 disease. There are currently no commercially available effective anti-viral therapies for COVID-19, urging the development of novel modalities. Here, we describe a molecular therapy specifically targeted to neutralize SARS-CoV-2, which consists of extracellular vesicles (EVs) containing a novel fusion tetraspanin protein, CD63, embedded within an anti-CoV-2 nanobody. These anti-CoV-2-enriched EVs bind SARS-CoV-2 spike protein at the receptor-binding domain (RBD) site and can functionally neutralize SARS-CoV-2. This work demonstrates an innovative EV-targeting platform that can be employed to target and inhibit the early stages of SARS-CoV-2 infection.

Keywords: CD63 fusion; COVID-19; SARS-CoV-2; VOC; extracellular vesicles; nanobody; neutralization; spike; therapeutic.

Graphical abstract

Figure 1

Characterization of targeted CD63 EVs (A) Schematic of the CD63 receptor and the insertion sites of the N6 scFv or VHH72 nanobody (Ex1.1, Ex2.2, Ex2.3, or Ex2.4) or a truncated CD4 domains 1 and 2 attached to the N terminus of CD63 (tCD4-D1D2). EC1 and EC2 denote the two main loops of CD63, and cysteine disulfide bonds are highlighted. A Nluc was fused in-frame to the C -terminus. (B) The N6-CD63 EVs were bound to beads and then incubated with gp120, and binding was assessed by flow cytometry, made relative to the CD63 control set at 100%. Error bars represent standard deviation generated from samples treated in triplicate. The p values were generated using a one-way ANOVA compared with the control (∗p < 0.05, ∗∗p < 0.01). (C) HEK293 cells that stably express gp160 were treated with N6-CD63 EVs, and the levels of Nluc were assessed at 18 h post-addition. The Nluc levels were normalized to HEK293 WT cells and made relative to the CD63 control set at 100%. Error bars represent standard deviation generated from samples treated in triplicate. The p values were generated using an unpaired Student’s t test compared with the control (∗∗p < 0.01). (D) TEM and (E) NTA analysis for the CD63 control and VHH72-CD63 EVs. (F) EVs and cell lysates were assessed by western blot for known EV markers (TSG101, ALIX, CD81) and the components of the CD63 fusion protein (Nluc and CD63). GAPDH was included as a loading control. Ladder molecular weights are indicated.

Figure 2

VHH72-CD63 EVs can bind SARS-CoV-2 spike (A) Beads were coated with an anti-SARS-CoV-2 spike antibody and bound to increasing concentrations of recombinant trimeric spike (0.1–10 μg/mL). The spike-bound beads were incubated with VHH72-CD63 EVs, and the levels of Nluc were assessed, which were made relative to the beads without spike, set at 100%. Error bars represent standard deviation generated from samples treated in duplicate. (B) The VHH72-CD63 EVs were bound to beads and incubated with SARS-CoV-2 RBD, and then flow cytometry was used to assess binding. Error bars represent standard deviation generated from samples treated in triplicate. The p values were generated using an unpaired Student’s t test compared with RBD-negative samples (∗p < 0.05). (C) TEM analysis of the VHH72-CD63 and CD63 containing EVs bound to gold-nanoparticle-labeled SARS-CoV-2 RBD. Red arrows highlight bound gold particles. Scale bar represents 200 nm. (D) HEK293 cells were transfected with a spike-expressing vector and treated with VHH72-CD63 EVs, and the levels of Nluc were assessed at 4 h post-addition. The Nluc levels were normalized to untransfected HEK293 cells and made relative to the CD63 control. Error bars represent standard deviation generated from samples treated in triplicate. The p values were generated using an unpaired Student’s t test compared with the control (∗∗∗p < 0.005). (E) A SARS-CoV-2 RBD fused to a HRP was pre-incubated with 2E9, 1E9, 5E8, and 1E8 total particles of the CD63 or VHH72-CD63 EVs, and the amount of RBD bound to ACE2 was assessed through a colorimetric assay. The percent inhibition was calculated from samples treated in duplicate. A recombinant VHH72 was included as a positive control.

Figure 3

VHH72-CD63 EVs broadly neutralize SARS-CoV-2 pseudovirus (A) The pseudotyped spike D614G-R682Q lentiviral particles were incubated with EVs and then transduced on HEK293-hACE2-hTMPRSS2 cells, and the levels of Fluc and Nluc were assessed at 72 h post-transduction. Error bars represent standard deviation generated from samples treated in triplicate. The p values were generated using an unpaired Student’s t -test compared with the CD63 control treated samples (∗∗p < 0.01). (B) The EVs were incubated at increasing concentrations with a set number of lentiviral particles (LP), and transductions were performed as described. Error bars represent standard deviation generated from samples treated in triplicate from two independent experiments. The p values were generated using a one-way ANOVA by comparing the means of the two experiments relative to the virus-only control (∗p < 0.05, ∗∗p < 0.01). (C) The pseudotyped lentiviral particles with the spike protein from the Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Epsilon (B.1.429) variants were incubated with 2E9 EVs, and then HEK293-hACE2-hTMPRSS2 cells were transduced. The levels of Fluc were assessed at 48 h post-transduction. The line represents the mean from samples treated in triplicate from two independent experiments. (D) The lentiviral particles pseudotyped with spikes from SARS-CoV-2 VOC were incubated with the S35 mAb (1.5 μg/mL) or CCP (P9K; 1:100 dilution), and then HEK293-hACE2-hTMPRSS2 cells were transduced. The line represents the mean from an experiment performed in triplicate. (E) VHH72-CD63 EVs were tested against lentivirus pseudotyped with the Delta (B.1.617.2) and Kappa (B.1.617.1) spike variants. The line represents the mean from samples treated in triplicate from two independent experiments, except for the S35- and recombinant VHH72-treated samples for the Kappa variant, which were generated from one independent experiment. The p values for (C), (D), and (E) were generated using a one-way ANOVA by comparing the means of the two experiments relative to the CD63 (C and E) or virus-only control (D) (∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Figure 4

VHH72-CD63 EVs neutralize SARS-CoV-2 (A–D) EVs were incubated at the described amounts with live SARS-CoV-2 from an ancestral strain (A) or VOC; Delta (B), Beta (C), Kappa (D), before infecting Vero E6 cells, and plaques were subsequently counted. A mAb targeted to the RBD, CB6, or 5309 were included as positive controls. Triplicate treated cells are shown with the standard error of the mean, and p values were determined by one-way ANOVA (Dunnett’s post-test) when compared against virus only (∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).