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Despite the introduction of several vaccines, COVID-19 continues to pose a serious health risk due to emerging variants of concern. DNA vaccines gained importance during the pandemic due to their advantages such as induction of both arms of immune response, rapid development, stability, and safety profiles. Here, we report the immunogenicity and protective efficacy of a DNA vaccine encoding spike protein with D614G mutation (named pcoSpikeD614G) and define a large-scale production process. According to the in vitro studies, pcoSpikeD614G expressed abundant spike protein in HEK293T cells. After the administration of pcoSpikeD614G to BALB\/c mice through intramuscular (IM) route and intradermal route using an electroporation device (ID\u2009+\u2009EP), it induced high level of anti-S1 IgG and neutralizing antibodies (<jats:italic>P<\/jats:italic>\u2009&lt;\u20090.0001), strong Th1-biased immune response as shown by IgG2a polarization (<jats:italic>P<\/jats:italic>\u2009&lt;\u20090.01), increase in IFN-\u03b3 levels (<jats:italic>P<\/jats:italic>\u2009&lt;\u20090.01), and increment in the ratio of IFN-\u03b3 secreting CD4<jats:sup>+<\/jats:sup> (3.78\u201310.19%) and CD8<jats:sup>+<\/jats:sup> (5.24\u201312.51%) T cells. Challenging K18-hACE2 transgenic mice showed that pcoSpikeD614G administered through IM and ID\u2009+\u2009EP routes conferred 90\u2013100% protection and there was no sign of pneumonia. Subsequently, pcoSpikeD614G was evaluated as a promising DNA vaccine candidate and scale-up studies were performed. Accordingly, a large-scale production process was described, including a 36\u00a0h fermentation process of <jats:italic>E. coli<\/jats:italic> DH5\u03b1 cells containing pcoSpikeD614G resulting in a wet cell weight of 242\u00a0g\/L and a three-step chromatography for purification of the pcoSpikeD614G DNA vaccine.<\/jats:p>","DOI":"10.1038\/s41598-024-64690-5","type":"journal-article","created":{"date-parts":[[2024,6,15]],"date-time":"2024-06-15T17:01:55Z","timestamp":1718470915000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Immunogenicity and protection efficacy of a COVID-19 DNA vaccine encoding spike protein with D614G mutation and optimization of large-scale DNA vaccine production"],"prefix":"10.1038","volume":"14","author":[{"given":"Ayt\u00fcl","family":"G\u00fcl","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sedef","family":"Erkunt 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