Neutralization of SARS-CoV-2 pseudovirus using ACE2-engineered extracellular vesicles


Posted: 2021-09-15 19:00:00
Acta Pharm Sin B . 2021 Sep 9. doi: 10.1016/j.apsb.2021.09.004. Online ahead of print. Affiliations Expand Affiliations 1 Artemisinin Research Center, First Clinical School, Guangzhou University of Chinese Medicine, Guangzhou 510450, China. 2 State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China. 3 Zhongshan Institute for Drug Discovery and Development, Chinese Academy of Sciences, Zhongshan 528437, China. 4 Center of Clinical Pharmacology, The Second Affiliated Hospital of Zhejiang University, School of Medicine, Hangzhou 310009, China. 5 NMPA Key Laboratory for Quality Research and Evaluation of Pharmaceutical Excipients, Shanghai 201203, China. 6 Taizhou University, School of Advanced Study, Institute of Natural Medicine and Health Product, Taizhou 318000, China. Item in Clipboard Canhao Wu et al. Acta Pharm Sin B. 2021. Show details Display options Display options Format Acta Pharm Sin B . 2021 Sep 9. doi: 10.1016/j.apsb.2021.09.004. Online ahead of print. Affiliations 1 Artemisinin Research Center, First Clinical School, Guangzhou University of Chinese Medicine, Guangzhou 510450, China. 2 State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China. 3 Zhongshan Institute for Drug Discovery and Development, Chinese Academy of Sciences, Zhongshan 528437, China. 4 Center of Clinical Pharmacology, The Second Affiliated Hospital of Zhejiang University, School of Medicine, Hangzhou 310009, China. 5 NMPA Key Laboratory for Quality Research and Evaluation of Pharmaceutical Excipients, Shanghai 201203, China. 6 Taizhou University, School of Advanced Study, Institute of Natural Medicine and Health Product, Taizhou 318000, China. Item in Clipboard CiteDisplay options Display options Format Abstract The spread of coronavirus disease 2019 (COVID-19) throughout the world has resulted in stressful healthcare burdens and global health crises. Developing an effective measure to protect people from infection is an urgent need. The blockage of interaction between angiotensin-converting enzyme 2 (ACE2) and S protein is considered an essential target for anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) drugs. A full-length ACE2 protein could be a potential drug to block early entry of SARS-CoV-2 into host cells. In this study, a therapeutic strategy was developed by using extracellular vesicles (EVs) with decoy receptor ACE2 for neutralization of SARS-CoV-2. The EVs embedded with engineered ACE2 (EVs-ACE2) were prepared; the EVs-ACE2 were derived from an engineered cell line with stable ACE2 expression. The potential effect of the EVs-ACE2 on anti-SARS-CoV-2 was demonstrated by both in vitro and in vivo neutralization experiments using the pseudovirus with the S protein (S-pseudovirus). EVs-ACE2 can inhibit the infection of S-pseudovirus in various cells, and importantly, the mice treated with intranasal administration of EVs-ACE2 can suppress the entry of S-pseudovirus into the mucosal epithelium. Therefore, the intranasal EVs-ACE2 could be a preventive medicine to protect from SARS-CoV-2 infection. This EVs-based strategy offers a potential route to COVID-19 drug development. Keywords: ACE2; ACE2, angiotensin-converting enzyme 2; BSA, bovine albumin; COVID-19; EVs, extracellular vesicles; Extracellular vesicles; FBS, fetal bovine serum; Intranasal administration; NTA, nanoparticle tracking analysis; Neutralization; PAGE, polyacrylamide gel electrophoresis; Pseudovirus; RIPA, radio immunoprecipitation assay; RLU, relative luminescence units; S protein, spike protein; SARS-CoV-2; SDS, sodium dodecyl sulfate; Spike protein; TEM, transmission electron microscope; WB, western blot. © 2021 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V. Conflict of interest statement The authors have no conflicts of interest to declare. Figures Graphical abstract Graphical abstract Graphical abstract Figure 1 (A) Schematic mechanism of EVs-ACE2… Figure 1 (A) Schematic mechanism of EVs-ACE2 inhibiting SARS-CoV-2 infection. The EVs-ACE2 were derived from… Figure 1 (A) Schematic mechanism of EVs-ACE2 inhibiting SARS-CoV-2 infection. The EVs-ACE2 were derived from the engineered HEK293T cells with stable ACE2 expression. EVs-ACE2 can competitively bind with the viruses via ACE2/S-protein interaction, thus blocking the virus to enter the host cells. (B) Size distributions of EVs-ACE2 measured by NTA. (C) Size distributions of EVs-control. (D) The median diameters of the EVs. (E) TEM images of EVs-ACE2. Scale bar = 50 nm. (F) ACE2 expression in EVs-ACE2 and EVs-Control. (G) The colloidal stability of the EVs. Data are presented as mean ± SD (n = 3); ns, no significance. Figure 2 Characterization of the S-pseudovirus. (A)… Figure 2 Characterization of the S-pseudovirus. (A) S-protein expression in the S-pseudovirus and EVs. (B)… Figure 2 Characterization of the S-pseudovirus. (A) S-protein expression in the S-pseudovirus and EVs. (B) Size distributions of S-pseudovirus determined by NTA. (C) The S-pseudovirus prepared with a packing ratio of pNL4-3.Luc.RE and nCOV.his-spike-FL of 1:0.5 (w/w) had the highest efficiency of cell entry. Scale bar = 100 μm. Figure 3 The EVs-ACE2 inhibited the pseudoviral… Figure 3 The EVs-ACE2 inhibited the pseudoviral infections in vivo . (A) WB of the… Figure 3 The EVs-ACE2 inhibited the pseudoviral infections in vivo. (A) WB of the ACE2 level in various cell lines. (B) Coomassie brilliant blue method: a standard protein human ACE2 was separately loaded in 0.025, 0.5, 0.75, 1.0, 1.5 μg to five lanes (lane 1-5); the whole EVs-ACE2 lysate was also separately loaded in 2.0, 5.0 μg to two lanes (lane 6&7). (C) EVs-ACE2 inhibited the cell entry of S-pseudovirus. Scale bars = 100 μm. (D) Flow cytometry assay results of (C). (E) EVs-ACE2 inhibited the infection of S-pseudovirus; RLU detected at 48 h after pseudoviral inoculation; scale bar = 100 μm. (F) The luciferase expression levels were qualified by qRT-PCR assay. Data are presented as mean ± SD (n = 3). **P < 0.01, ***P < 0.001, ****P<0.0001; ns, no significance. Figure 4 In vivo neutralization test. (A)… Figure 4 In vivo neutralization test. (A) ACE2 expression in the murine nasal mucosa. (B)… Figure 4 In vivo neutralization test. (A) ACE2 expression in the murine nasal mucosa. (B) The quantitative analysis of the overlap proportion in the images of (C) using ImageJ. (C) Fluorescence images of the nasal mucosa cryosection slices from the mice challenged by the S-pseudovirus with the DiO-labeled EVs-ACE2/EVs-Control pretreatment. Scale bars = 100 μm. (D) The luciferase expression levels of nasal mucosa cryosection tissues were qualified by qRT-PCR assay (n = 3). (E) Flow cytometry assay of nasal mucosal tissues after S-pseudovirus challenge. Data are presented as mean ± SD (n = 3). **P < 0.01, ns, no significance. Figure 5 (A) Hematoxylin and eosin staining… Figure 5 (A) Hematoxylin and eosin staining of different sections taken 3 days after nasal… Figure 5 (A) Hematoxylin and eosin staining of different sections taken 3 days after nasal administration of the EVs. Scale bars = 50 μm. (B) Serum chemistry test. TP, total protein; ALB, albumin; ALT, alanine aminotransferase; Urea, urea nitrogen; CRE, creatinine; Ca, calcium; P, phosphorus; K, potassium; and Na, sodium. Data are presented as mean ± SD (n = 3); ns, no significance. References Covid-19 coronavirus pandemic. Available from: https://www.worldometers.info/coronavirus/(Accessed May 6, 2021). Weng J., Li Y., Li J., Shen L., Zhu L., Liang Y. Gastrointestinal sequelae 90 days after discharge for COVID-19. Lancet Gastroenterol Hepatol. 2021;6:344–346. - PMC - PubMed Huang C., Huang L., Wang Y., Li X., Ren L., Gu X. 6-Month consequences of COVID-19 in patients discharged from hospital: a cohort study. Lancet. 2021;397:220–232. - PMC - PubMed Wang Y., Zhang D., Du G., Du R., Zhao J., Jin Y. Remdesivir in adults with severe COVID-19: a randomised, double-blind, placebo-controlled, multicentre trial. Lancet. 2020;395:1569–1578. - PMC - PubMed Singh B., Ryan H., Kredo T., Chaplin M., Fletcher T. Chloroquine or hydroxychloroquine for prevention and treatment of COVID-19. Cochrane Database Syst Rev. 2021;2 CD013587. - PMC - PubMed Show all 45 references

参考サイト PubMed: exsome


Powered by Stromvergleich


バイオクイックニュース日本語版:エクソソーム特集

バイオクイックニュース日本語版
10月 16, 2019 バイオアソシエイツ

南カリフォルニアのHOAG 病院は、癌の遺伝的リスクが高い人の癌検出と管理の早期疾患マーカーとしてエクソソームを研究。

カリフォルニア州オレンジ郡にある非営利の地域医療提供ネットワークのHoag Memorial Hospital Presbyterianは、癌診断、癌の進行、および治療抵抗性の初期疾患マーカーの可能性を特定および特徴付ける研究の開始を発表した。 Exosome Sciences社および、Aethlon Medical社の子会社との提携により、癌の遺伝的リスクが高い癌患者の エクソソーム 研究を開始する。…

ゲスト 378人 と メンバー1人 がオンラインです