{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,19]],"date-time":"2026-02-19T02:57:32Z","timestamp":1771469852031,"version":"3.50.1"},"reference-count":45,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2022,4,13]],"date-time":"2022-04-13T00:00:00Z","timestamp":1649808000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000780","name":"European Union","doi-asserted-by":"publisher","award":["730964"],"award-info":[{"award-number":["730964"]}],"id":[{"id":"10.13039\/501100000780","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia","doi-asserted-by":"publisher","award":["UIDB\/04462\/2020"],"award-info":[{"award-number":["UIDB\/04462\/2020"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia","doi-asserted-by":"publisher","award":["UIDP\/04462\/2020"],"award-info":[{"award-number":["UIDP\/04462\/2020"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia","doi-asserted-by":"publisher","award":["IF\/01704\/2014"],"award-info":[{"award-number":["IF\/01704\/2014"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia","doi-asserted-by":"publisher","award":["EXPL\/BBB-BIO\/1541\/2013"],"award-info":[{"award-number":["EXPL\/BBB-BIO\/1541\/2013"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia","doi-asserted-by":"publisher","award":["IF\/01704\/2014\/CP1229\/CT0001"],"award-info":[{"award-number":["IF\/01704\/2014\/CP1229\/CT0001"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia","doi-asserted-by":"publisher","award":["SFRH\/BD\/138937\/2018"],"award-info":[{"award-number":["SFRH\/BD\/138937\/2018"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) homotrimeric spike (S) protein is responsible for mediating host cell entry by binding to the angiotensin-converting enzyme 2 (ACE2) receptor, thus being a key viral antigen to target in a coronavirus disease 19 (COVID-19) vaccine. Despite the availability of COVID-19 vaccines, low vaccine coverage as well as unvaccinated and immune compromised subjects are contributing to the emergence of SARS-CoV-2 variants of concern. Therefore, continued development of novel and\/or updated vaccines is essential for protecting against such new variants. In this study, we developed a scalable bioprocess using the insect cells-baculovirus expression vector system (IC-BEVS) to produce high-quality S protein, stabilized in its pre-fusion conformation, for inclusion in a virosome-based COVID-19 vaccine candidate. By exploring different bioprocess engineering strategies (i.e., signal peptides, baculovirus transfer vectors, cell lines, infection strategies and formulation buffers), we were able to obtain ~4 mg\/L of purified S protein, which, to the best of our knowledge, is the highest value achieved to date using insect cells. In addition, the insect cell-derived S protein exhibited glycan processing similar to mammalian cells and mid-term stability upon storage (up to 90 days at \u221280 and 4 \u00b0C or after 5 freeze-thaw cycles). Noteworthy, antigenicity of S protein, either as single antigen or displayed on the surface of virosomes, was confirmed by ELISA, with binding of ACE2 receptor, pan-SARS antibody CR3022 and neutralizing antibodies to the various epitope clusters on the S protein. Binding capacity was also maintained on virosomes-S stored at 4 \u00b0C for 1 month. This work demonstrates the potential of using IC-BEVS to produce the highly glycosylated and complex S protein, without compromising its integrity and antigenicity, to be included in a virosome-based COVID-19 vaccine candidate.<\/jats:p>","DOI":"10.3390\/pharmaceutics14040854","type":"journal-article","created":{"date-parts":[[2022,4,13]],"date-time":"2022-04-13T23:07:16Z","timestamp":1649891236000},"page":"854","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":23,"title":["Insect Cells for High-Yield Production of SARS-CoV-2 Spike Protein: Building a Virosome-Based COVID-19 Vaccine Candidate"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1964-1599","authenticated-orcid":false,"given":"B\u00e1rbara","family":"Fernandes","sequence":"first","affiliation":[{"name":"iBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2780-901 Oeiras, Portugal"},{"name":"ITQB NOVA, Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Av. da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0899-0649","authenticated-orcid":false,"given":"Rute","family":"Castro","sequence":"additional","affiliation":[{"name":"iBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2780-901 Oeiras, Portugal"}]},{"given":"Farien","family":"Bhoelan","sequence":"additional","affiliation":[{"name":"Mymetics BV, J.H. Oortweg 21, 2333 CH Leiden, The Netherlands"}]},{"given":"Denzel","family":"Bemelman","sequence":"additional","affiliation":[{"name":"Mymetics BV, J.H. Oortweg 21, 2333 CH Leiden, The Netherlands"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7155-0059","authenticated-orcid":false,"given":"Ricardo A.","family":"Gomes","sequence":"additional","affiliation":[{"name":"iBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2780-901 Oeiras, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7782-6319","authenticated-orcid":false,"given":"J\u00falia","family":"Costa","sequence":"additional","affiliation":[{"name":"ITQB NOVA, Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Av. da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7245-6785","authenticated-orcid":false,"given":"Patr\u00edcia","family":"Gomes-Alves","sequence":"additional","affiliation":[{"name":"iBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2780-901 Oeiras, Portugal"},{"name":"ITQB NOVA, Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Av. da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"given":"Toon","family":"Stegmann","sequence":"additional","affiliation":[{"name":"Mymetics BV, J.H. Oortweg 21, 2333 CH Leiden, The Netherlands"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2574-4092","authenticated-orcid":false,"given":"Mario","family":"Amacker","sequence":"additional","affiliation":[{"name":"Mymetics SA, Route de la Corniche 4, 1066 Epalinges, Switzerland"},{"name":"Department of Biomedical Research (DBMR), Department of Pulmonary Medicine, Bern University Hospital, University of Bern, 3008 Bern, Switzerland"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1445-3556","authenticated-orcid":false,"given":"Paula M.","family":"Alves","sequence":"additional","affiliation":[{"name":"iBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2780-901 Oeiras, Portugal"},{"name":"ITQB NOVA, Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Av. da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]},{"given":"Sylvain","family":"Fleury","sequence":"additional","affiliation":[{"name":"Mymetics SA, Route de la Corniche 4, 1066 Epalinges, Switzerland"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0414-4899","authenticated-orcid":false,"given":"Ant\u00f3nio","family":"Rold\u00e3o","sequence":"additional","affiliation":[{"name":"iBET, Instituto de Biologia Experimental e Tecnol\u00f3gica, Apartado 12, 2780-901 Oeiras, Portugal"},{"name":"ITQB NOVA, Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa, Av. da Rep\u00fablica, 2780-157 Oeiras, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2022,4,13]]},"reference":[{"key":"ref_1","unstructured":"(2021, August 11). 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