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.

Introduction

Despite the tremendous success of the COVID-19 vaccine development, the pandemic caused by SARS-CoV-2 has been challenging due to fast-evolving mutant strains^1,2,3,4,5 and slow vaccination globally. Shortly after the outbreak, over 1000 SARS-CoV-2 variants were detected⁶ and several mutant strains (α, β, and δ) dominated with higher infection rates and/or fatality than the wild-type (WT) strain^4,5,7,8. Fully vaccinated populations only reached below 30% worldwide and about half in the US as of September 2021. Moreover, the risk of future emerging coronaviruses infecting human always exist. To better protect vulnerable people, both unvaccinated and vaccinated, it is urgent to develop novel therapeutics that can broadly target distinct strains of evolving SARS-CoV-2 and future coronaviruses.

Similar to other coronaviruses such as SARS-CoV, which caused an outbreak in 2003⁹, the WT and mutant strains of SARS-CoV-2 infect host cells such as human pneumocytes via the entry receptor angiotensin-converting enzyme 2 (ACE2)^1,10,11,12. The mutations-caused alterations in the viral proteins such as the attachment protein—spike glycoprotein (S), in particular the external receptor-binding domain (RBD) in the variants (α, β, and δ), render a greater binding affinity than the WT in binding to ACE2^{1,4,5,10,11,12}. Approaches to block or impede the viral interaction with the entry receptor ACE2 on the host cell, including S-specific neutralization antibodies (Abs)^{13,14,15,16,17,18,19,20,21,22,23,24,25} and rhACE2^{26,27,28,29,30}, inhibit infectivity and prevent COVID-19. Although many high-affinity monoclonal antibodies were identified from convalescent patients and engineered as therapeutics to treat mild diseases of COVID-19^{13,14,15,16,17,18,19,20,21,22,23,24,25}, many did not show favorable efficacy for hospitalized patients^31,32 and some of those with emergency use authorization (EUA) lost efficacy against new variants, such as δ³³. Monoclonal antibodies targeting specific epitopes of SARS-CoV-2 antigens appear to have limited capacity to broadly neutralize current and future mutant strains^4,5,6. Nonetheless, since the plasma or sera of convalescent COVID-19 patients have reportedly been used to treat active infection of SARS-CoV-2 or severe diseases^34,35, we aimed to identify previously unknown anti-viral components from the human plasma that may inform on potential new therapeutics.

Extracellular vesicles (EVs) are one of the essential components of liquid biopsy such as blood, including large microvesicles (200–1000 µm), small exosomes (50–200 µm), and newly identified exomeres (<50 µm)^36,37. Exosomes are amongst the best characterized small EVs that likely participate in a variety of physiological and pathobiological functions^{38,39,40,41,42} as well as serve as novel biomarkers and next-generation biologic therapeutics^43,44. They present many proteins on the surface reminiscent of their cellular counterpart, such as immune regulators of myeloid and lymphoid cells to affect antiviral immune response^42,45,46. Exosomes derived from both plants and human specimens have been used in clinical trials to treat inflammatory diseases and cancers^47,48,49. In line with widely adopted nomenclature in the EV field³⁷ and the possibility that heterogenous vesicle populations may be isolated, we collectively refer to the enriched exosomes therein as ‘EVs’.

Here we detected a significant increase in circulating ACE2⁺ EVs in the plasma of COVID-19 patients, in particular during the acute phase. Importantly, ACE2⁺ EVs (evACE2) isolated from engineered cell lines inhibit SARS-CoV-2 infection by blocking the viral spike protein binding with its cellular receptor ACE2 in host cells. Our observations demonstrate that evACE2 is a decoy antiviral mechanism to prevent SARS-CoV-2 infection, thus providing a rationale for the use of evACE2 to combat COVID-19.

Results

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

We previously established an automated and high throughput method, microflow vesiclometry (MFV), to detect and profile the surface proteins of blood EVs at single-particle resolution⁴³. Direct MFV analysis of circulating EVs in human plasma samples (Table 1, N = 89) revealed elevated ACE2⁺ EVs in the plasma of COVID-19 patients in comparison to seronegative controls, with a more dramatic increase in the acute phase and a modest elevation in the convalescent-phase (Fig. 1a, b, Supplementary Fig. 1a–d), with the latter in association with COVID-19 severe disease showing relatively higher levels in inpatient samples (Supplementary Fig. 1b). ACE2⁺ EVs were enriched in CD63⁺ EV subsets from COVID-19 patients (Fig. 1c). Consistently, SARS-CoV-2 infection triggered secretion of ACE2⁺TSG101⁺ EVs by human pneumocyte A549 cells overexpressing ACE2 (Supplementary Fig. 1e), implying that upregulated production of ACE2⁺ EVs is part of the innate response to SARS-CoV-2 infection in COVID-19 patients.