Quantitative proteomics and biological activity of extracellular vesicles engineered to express SARS‐CoV‐2 spike protein

Abstract

SARS-CoV-2 viral infection led to the devastating COVID-19 pandemic, where illness stemmed from interactions between virions and recipient host cells resulting in multi-layered pathological consequences. The role of the infection portal is now understood to be the cellular angiotensin converting enzyme-2 (ACE2) receptor, which binds to viral spike (S) protein initiating virion internalisation process. Since SARS-CoV-2 virions bear some resemblance to endogenously produced small extracellular vesicles (sEVs) we reasoned that EVs engineered to express S protein (viral mimics) may interfere with viral infection. Here, we report generation of HEK293T cells producing sEVs enriched for transmembrane S-protein tagged with green fluorescent protein (S/GFP). Strikingly, S protein drove the GFP tag to the membrane of sEVs, while GFP alone was not efficiently included in the sEV cargo. Highthroughput quantitative proteomics revealed that S/GFP sEVs contained over 1000 proteins including canonical components of the exosomal pathway such as ALIX, syntenin-1, and tetraspanins (CD81, CD9), but depleted for calnexin and cytochrome c. We found that 84 sEV proteins were significantly altered by the presence of S/GFP. S protein expressing EVs efficiently adhered to target cells in an ACE2-dependent manner, but they were poorly internalised. Importantly, prolonged administration of S/GFP EV to K18-hACE2 mice provided a significant protection against SARS-CoV-2 infection. Thus, the generation of sEV containing S protein can be considered as a novel therapeutic approach in reducing the transmission of SARS-CoV-2.

Scalable purification of extracellular vesicles with high yield and purity using multimodal flowthrough chromatography

“…However, more recent studies that have separated cell derived particle preparations into their respective constituents using density gradient ultracentrifugation have detected calnexin in EV associated fractions, including in particle preparations derived from HEK293T cells. These studies have shown that calnexin is not as underrepresented as other non‐EV proteins more usually associated with nuclear and mitochondrial processes and post‐translational modification (Choi et al., 2022 ; Kugeratski et al., 2021 ). Furthermore, calnexin has also been suggested as a marker of large EVs (>150 nm) (Théry et al., 2018 ).…”

Immunomodulatory properties of Leishmania tarentolae extracellular vesicles containing the Spike protein of SARS-CoV-2

“…Future studies need to evaluate the adjuvant potential of EV-rS by immunizing mice and to investigate their ability to elicit neutralizing antibodies towards SARS-CoV-2. In this regard, the use of EVs or exosomes as a vehicle to deliver SARS-CoV-2 Spike for vaccination purposes has been proposed and tested in other systems (Choi et al, 2022;Jiang et al, 2022;Cacciottolo et al, 2023a;Cacciottolo et al, 2023b;Kim and Thapa, 2023). In one of those studies, Cacciottolo and colleagues (Cacciottolo et al, 2023a) have expressed the Delta variant Spike linked to the N-terminal of the exosome-specific tetraspanin CD9 by a synthetic transmembrane domain and a secretion signal peptide.…”

Extracellular vesicles and angiotensin-converting enzyme 2 in COVID-19 disease

“…Previous studies have shown that CD9 may collaborate with TMPRSS2 in cleaving viral fusion glycoproteins and facilitate quick entry of coronavirus (e.g., MERS-CoV) into lung cells [54]. Studies also showed that membrane surface CD9 may also be involved in entry and exit mechanisms in respiratory viruses, including SARS-CoV-2 virus [48,[55][56][57].…”