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The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major target for the COVID-19 vaccines. Among the available vaccine types, DNA vaccines are a promising alternative to conventional vaccines. Here, we developed a novel DNA vaccine containing the SARS-CoV-2 S1 subunit and an optimized flagellin adjuvant (FliC\u0394D2D3) from\n            <jats:italic>Salmonella typhimurium<\/jats:italic>\n            . The S1 gene and S1-\n            <jats:italic>fliC<\/jats:italic>\n            \u0394D2D3 fusion gene were codon optimized and then cloned into the antibiotic-resistance-gene-free vector\n            <jats:italic>asd<\/jats:italic>\n            -pVAX1, producing\n            <jats:italic>asd<\/jats:italic>\n            -pVAX1-S1 and\n            <jats:italic>asd<\/jats:italic>\n            -pVAX1-S1-\n            <jats:italic>fliC<\/jats:italic>\n            \u0394D2D3, respectively. The expression of S1 and S1-FliC\u0394D2D3 proteins was confirmed after transient expression in HEK293T cells. After\n            <jats:italic>in vitro<\/jats:italic>\n            characterization of the vaccine candidates, we evaluated the humoral and cellular immune responses induced in BALB\/c mice. The DNA vaccine induced a long-lasting humoral response for approximately 4 months, and the elicited serum antibodies efficiently blocked the binding of the SARS-CoV-2 S1 receptor-binding domain to its entry receptor (angiotensin-converting enzyme 2). Moreover, the DNA vaccine induced strong Th1\/Th2-mixed immune responses, as demonstrated by the similar levels of S1-specific immunoglobulin (Ig) G1 and IgG2a in the serum and the significantly elevated Th1-type (IFN-\u03b3 and TNF-\u03b1) and Th2-type (IL-4 and IL-6) cytokine expression from splenic lymphocytes. Importantly,\n            <jats:italic>asd<\/jats:italic>\n            -pVAX1-S1-\n            <jats:italic>fliC<\/jats:italic>\n            \u0394D2D3 immunization in mice could significantly enhance the levels of immune responses compared to\n            <jats:italic>asd<\/jats:italic>\n            -pVAX1-S1. This study provides crucial information regarding the selection of a safer DNA vector and a new adjuvant for the vaccine development of SARS-CoV-2 and other infectious diseases.\n          <\/jats:p>\n          <jats:sec>\n            <jats:title>IMPORTANCE<\/jats:title>\n            <jats:p>The development of safe and effective vaccines is needed to control the transmission of coronavirus disease 2019 (COVID-19). Synthetic DNA vaccines represent a promising platform in response to such outbreaks. Here, DNA vaccine candidates were developed using an optimized antibiotic-resistance gene-free asd-pVAX1 vector. An optimized flagellin (FliC) adjuvant was designed by fusion expression to increase the immunogenicity of the S1 antigen. S1 and S1-FliC\u0394D2D3 proteins were strongly expressed in mammalian cells. The FliC\u0394D2D3-adjuvanted DNA vaccine induced Th1\/Th2-mixed immune responses and high titers of neutralizing antibodies. This study provides crucial information regarding the selection of a safer DNA vector and adjuvant for vaccine development. Our FliC\u0394D2D3-adjuvanted S1 DNA vaccine is more potent at inducing both humoral and cellular immune responses than S1 alone. This finding provides a new idea for the development of novel DNA vaccines against COVID-19 and could be further applied for the development of other vaccines.<\/jats:p>\n          <\/jats:sec>","DOI":"10.1128\/spectrum.02549-23","type":"journal-article","created":{"date-parts":[[2023,11,1]],"date-time":"2023-11-01T13:21:51Z","timestamp":1698844911000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":8,"title":["Enhanced immunogenicity elicited by a novel DNA vaccine encoding the SARS-CoV-2 S1 protein fused to the optimized flagellin of\n            <i>Salmonella typhimurium<\/i>\n            in mice"],"prefix":"10.1128","volume":"11","author":[{"given":"Li","family":"Song","sequence":"first","affiliation":[{"name":"Jiangsu Key Laboratory of Zoonosis, Yangzhou University , Yangzhou, Jiangsu, China"},{"name":"Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University , Yangzhou, Jiangsu, China"},{"name":"Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agrifood Safety and Quality, Ministry of Agriculture of China, Yangzhou University , Yangzhou, Jiangsu, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qiaoju","family":"Wang","sequence":"additional","affiliation":[{"name":"Jiangsu Key Laboratory of Zoonosis, Yangzhou University , Yangzhou, Jiangsu, China"},{"name":"Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University , Yangzhou, Jiangsu, China"},{"name":"Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agrifood Safety and Quality, Ministry of Agriculture of China, Yangzhou University , Yangzhou, Jiangsu, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yaya","family":"Wen","sequence":"additional","affiliation":[{"name":"Jiangsu Key Laboratory of Zoonosis, Yangzhou University , Yangzhou, Jiangsu, China"},{"name":"Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University , Yangzhou, Jiangsu, China"},{"name":"Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agrifood Safety and Quality, Ministry of Agriculture of China, Yangzhou University , Yangzhou, Jiangsu, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruimeng","family":"Tan","sequence":"additional","affiliation":[{"name":"Jiangsu Key Laboratory of Zoonosis, Yangzhou University , Yangzhou, Jiangsu, China"},{"name":"Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University , Yangzhou, Jiangsu, China"},{"name":"Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agrifood Safety and Quality, Ministry of Agriculture of 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Wei Sheng Wu Xue Bao 45:937\u2013941.","journal-title":"Wei Sheng Wu Xue Bao"},{"key":"e_1_3_2_50_2","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2017.00326"}],"container-title":["Microbiology Spectrum"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/spectrum.02549-23","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/spectrum.02549-23","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,12,12]],"date-time":"2023-12-12T13:21:03Z","timestamp":1702387263000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.asm.org\/doi\/10.1128\/spectrum.02549-23"}},"subtitle":[],"editor":[{"given":"Daniela S.","family":"Rajao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2023,12,12]]},"references-count":49,"journal-issue":{"issue":"6","published-print":{"date-parts":[[2023,12,12]]}},"alternative-id":["10.1128\/spectrum.02549-23"],"URL":"https:\/\/doi.org\/10.1128\/spectrum.02549-23","relation":{},"ISSN":["2165-0497"],"issn-type":[{"value":"2165-0497","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,12,12]]},"assertion":[{"value":"2023-06-18","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2023-09-08","order":1,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2023-11-01","order":2,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e02549-23"}}