{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T00:54:05Z","timestamp":1760057645580,"version":"build-2065373602"},"reference-count":54,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2025,2,17]],"date-time":"2025-02-17T00:00:00Z","timestamp":1739750400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"NSERC (Natural Sciences and Engineering Research Council of Canada)","award":["RGPIN #388697","#ALLRP 550110-20","SEMUMS. REC.1399.174"],"award-info":[{"award-number":["RGPIN #388697","#ALLRP 550110-20","SEMUMS. REC.1399.174"]}]},{"name":"NSERC Alliance COVID-19","award":["RGPIN #388697","#ALLRP 550110-20","SEMUMS. REC.1399.174"],"award-info":[{"award-number":["RGPIN #388697","#ALLRP 550110-20","SEMUMS. REC.1399.174"]}]},{"name":"Semnan University of Medical Sciences, Semnan, Iran","award":["RGPIN #388697","#ALLRP 550110-20","SEMUMS. REC.1399.174"],"award-info":[{"award-number":["RGPIN #388697","#ALLRP 550110-20","SEMUMS. REC.1399.174"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Background\/Objectives: The SARS-CoV-2\u2019s high mutations and replication rates contribute to its high infectivity and resistance to current vaccinations and treatments. The primary cause of resistance to most current treatments aligns within the coding regions for the spike S protein of SARS-CoV-2 that has mutated. As a potential novel immunotherapy, we generated a novel fusion protein composed of a soluble ACE2 (sACE2) linked to llama-derived anti-CD16 that targets different variants of spike proteins and enhances natural killer cells to target infected cells. Methods: Here, we generated a novel sACE2-AntiCD16VHH fusion protein using a Gly4Ser linker, synthesized and cloned into the pLVX-EF1alpha-IRES-Puro vector, and further expressed in ExpiCHO-S cells and purified using Ni+NTA chromatography. Results: The fusion protein significantly blocked SARS-CoV-2 alpha, beta, delta, gamma, and omicron S-proteins binding and activating angiotensin-converting enzyme receptor-2 (ACE2) on ACE2-expressing RAW-Blue macrophage cells and the secretion of several key inflammatory cytokines, G-CSF, MIP-1A, and MCP-1, implicated in the cytokine release storm (CRS). The sACE2-Anti-CD16VHH fusion protein also bridged NK cells to ACE2-expressing human lung carcinoma A549 cells and significantly activated NK-dependent cytotoxicity. Conclusions: The findings show that a VHH directed against CD16 could be an excellent candidate to be linked to soluble ACE2 to generate a bi-specific molecule (sACE2-AntiCD16VHH) suitable for bridging effector cells and infected target cells to inhibit SARS-CoV-2 variant spike proteins binding to the ACE2 receptor in the RAW-Blue cell line and pro-inflammatory cytokines and to activate natural killer cell cytotoxicity.<\/jats:p>","DOI":"10.3390\/vaccines13020199","type":"journal-article","created":{"date-parts":[[2025,2,17]],"date-time":"2025-02-17T07:48:22Z","timestamp":1739778502000},"page":"199","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Novel sACE2-Anti-CD16VHH Fusion Protein Surreptitiously Inhibits SARS-CoV-2 Variant Spike Proteins and Macrophage Cytokines, and Activates Natural Killer Cell Cytotoxicity"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3559-5548","authenticated-orcid":false,"given":"Abdolkarim","family":"Sheikhi","sequence":"first","affiliation":[{"name":"Department of Biomedical & Molecular Sciences, Queen\u2019s University, Kingston, ON K7L 3N6, Canada"},{"name":"Department of Immunology, School of Medicine, Dezful University of Medical Sciences, Dezful 64616-43993, Iran"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-7135-2570","authenticated-orcid":false,"given":"Leili","family":"Baghaie","sequence":"additional","affiliation":[{"name":"Department of Biomedical & Molecular Sciences, Queen\u2019s University, Kingston, ON K7L 3N6, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fatemeh","family":"Rahbarizadeh","sequence":"additional","affiliation":[{"name":"Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran 14115-331, Iran"},{"name":"Research and Development Center of Biotechnology, Tarbiat Modares University, Tehran 14115-331, Iran"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9901-7851","authenticated-orcid":false,"given":"Pooria","family":"Safarzadeh Kozani","sequence":"additional","affiliation":[{"name":"Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran 14115-331, Iran"},{"name":"Research and Development Center of Biotechnology, Tarbiat Modares University, Tehran 14115-331, Iran"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3948-4642","authenticated-orcid":false,"given":"Cobra","family":"Moradian","sequence":"additional","affiliation":[{"name":"Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran 14115-331, Iran"},{"name":"Research and Development Center of Biotechnology, Tarbiat Modares University, Tehran 14115-331, Iran"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mohammadreza","family":"Davidi","sequence":"additional","affiliation":[{"name":"Faculty of Medicine, Kazeroon Azad University, Kazeroon 14778-93855, Iran"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Narges","family":"Baharifar","sequence":"additional","affiliation":[{"name":"Department of Immunology, School of Medicine, Dezful University of Medical Sciences, Dezful 64616-43993, Iran"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Golnaz","family":"Kaboli","sequence":"additional","affiliation":[{"name":"Department of Immunology, School of Medicine, Dezful University of Medical Sciences, Dezful 64616-43993, Iran"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mehdi","family":"Sheikhi","sequence":"additional","affiliation":[{"name":"Faculty of Medicine, Kazeroon Azad University, Kazeroon 14778-93855, Iran"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yunfan","family":"Li","sequence":"additional","affiliation":[{"name":"Faculty of Arts and Science, Queen\u2019s University, Kingston, ON K7L 3N9, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-6322-3162","authenticated-orcid":false,"given":"Mohammadamin","family":"Meghdadi","sequence":"additional","affiliation":[{"name":"Faculty of Health Sciences, Queen\u2019s University, Kingston, ON K7L 3N9, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-0220-3211","authenticated-orcid":false,"given":"Abdulrahman M.","family":"Yaish","sequence":"additional","affiliation":[{"name":"Faculty of Health Sciences, Queen\u2019s University, Kingston, ON K7L 3N9, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-5474-9459","authenticated-orcid":false,"given":"Aiden H.","family":"Yu","sequence":"additional","affiliation":[{"name":"Faculty of Health Sciences, Queen\u2019s University, Kingston, ON K7L 3N9, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"William W.","family":"Harless","sequence":"additional","affiliation":[{"name":"ENCYT Technologies Inc., Membertou, NS B1S 0H1, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8471-5481","authenticated-orcid":false,"given":"Myron R.","family":"Szewczuk","sequence":"additional","affiliation":[{"name":"Department of Biomedical & Molecular Sciences, Queen\u2019s University, Kingston, ON K7L 3N6, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,2,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"8801","DOI":"10.1128\/JVI.77.16.8801-8811.2003","article-title":"The coronavirus spike protein is a class I virus fusion protein: 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