A lipid nanoparticle (LNP)-based DNA vaccine, LNP-HPS, was developed to deliver plasmid DNA encoding the HexaPro version of the SARS-CoV-2 spike protein. This vaccine was tested in female preclinical models and showed robust protection against SARS-CoV-2 variants, including Gamma (P.1) and Omicron (B.1.1.529). The LNP-HPS vaccine elicited strong humoral and T cell immune responses, generating high levels of neutralizing antibodies against both variants. The vaccine demonstrated protective efficacy comparable to the Biontech/Pfizer mRNA vaccine in animal models, with reduced viral load, lower lethality, and less lung damage in vaccinated mice and hamsters. The LNP-HPS vaccine was formulated using a library of 15 engineered LNPs, each encapsulating unique barcoded DNA, to identify the most effective delivery system. The lead LNP, B4, was selected for its efficient delivery to antigen-presenting cells and minimal delivery to the heart and liver. The LNP-HPS vaccine showed enhanced transfection efficiency in various cell types and was effective in inducing protective immunity against SARS-CoV-2 variants. The vaccine also induced strong CD4+ and CD8+ T cell responses, with increased production of IFN-γ and granzyme-B. The LNP-HPS vaccine demonstrated comparable immunogenicity and protective efficacy to the Biontech/Pfizer vaccine, highlighting its potential as a candidate for combating SARS-CoV-2 and its variants. The study underscores the effectiveness of LNP-based DNA vaccines in eliciting robust immune responses and provides a promising platform for future vaccine development.A lipid nanoparticle (LNP)-based DNA vaccine, LNP-HPS, was developed to deliver plasmid DNA encoding the HexaPro version of the SARS-CoV-2 spike protein. This vaccine was tested in female preclinical models and showed robust protection against SARS-CoV-2 variants, including Gamma (P.1) and Omicron (B.1.1.529). The LNP-HPS vaccine elicited strong humoral and T cell immune responses, generating high levels of neutralizing antibodies against both variants. The vaccine demonstrated protective efficacy comparable to the Biontech/Pfizer mRNA vaccine in animal models, with reduced viral load, lower lethality, and less lung damage in vaccinated mice and hamsters. The LNP-HPS vaccine was formulated using a library of 15 engineered LNPs, each encapsulating unique barcoded DNA, to identify the most effective delivery system. The lead LNP, B4, was selected for its efficient delivery to antigen-presenting cells and minimal delivery to the heart and liver. The LNP-HPS vaccine showed enhanced transfection efficiency in various cell types and was effective in inducing protective immunity against SARS-CoV-2 variants. The vaccine also induced strong CD4+ and CD8+ T cell responses, with increased production of IFN-γ and granzyme-B. The LNP-HPS vaccine demonstrated comparable immunogenicity and protective efficacy to the Biontech/Pfizer vaccine, highlighting its potential as a candidate for combating SARS-CoV-2 and its variants. The study underscores the effectiveness of LNP-based DNA vaccines in eliciting robust immune responses and provides a promising platform for future vaccine development.