Linear DNA expression cassette encapsulated in lipid nanoparticles confers complete protection against influenza A virus in mice

DNA-based vaccines have been developed and tested against a range of infectious agents, including influenza A viruses (IAV). Bacterial plasmid DNA is widely used as a vector for delivering genes encoding vaccine immunogens. However, the inclusion of antibiotic resistance genes in conventional plasmid constructs raises safety concerns. Linear DNA expression cassettes produced via PCR that lack antibiotic resistance genes, and bacterial sequences can serve as a safer alternative vector. In this study, we employed a mouse model of IAV to evaluate the protective efficacy of a lipid nanoparticle (LNP)-encapsulated linear DNA expression cassette containing the hemagglutinin gene of IAV H1N1 produced by PCR amplification. For comparison, a circular plasmid DNA containing the same gene was also constructed. The LNP-encapsulated DNA cassette (LNP-L) exhibited similar physicochemical characteristics, such as particle size, polydispersity index, zeta potential, and encapsulation efficiency, compared to the LNP-encapsulated circular plasmid DNA (LNP-P). However, LNP-L produced significantly lower transgene expression than LNP-P in cell culture. Mice immunized with LNP-L developed a delayed onset of antibody response compared to those immunized with LNP-P, but the magnitudes of both humoral and cell-mediated immune responses were comparable between the two groups after booster immunization. Moreover, mice immunized with LNP-L exhibited both Th1 and Th2 responses, similar to those immunized with LNP-P. Importantly, mice immunized with either LNP-L or LNP-P were fully protected upon challenge with a lethal dose of IAV H1N1. These findings suggest that linear DNA expression cassettes encapsulated in LNPs represent a promising strategy for vaccine development.IMPORTANCELipid nanoparticle (LNP)-encapsulated DNA vaccines represent a promising platform for the rapid development of vaccines against infectious pathogens. However, the inclusion of an antibiotic-resistance gene in plasmid DNA vectors used to deliver vaccine immunogens raises potential safety concerns. This study explores the feasibility of using a linear expression cassette produced via PCR amplification as an alternative system for delivering vaccine immunogens. Compared with conventional plasmid DNA, the linear expression cassette contains only essential elements, including a promoter, the gene encoding the vaccine immunogen, and a polyadenylation signal, and can be produced in a cell-free system. Using a mouse model for influenza A virus, we demonstrate that an LNP-encapsulated linear DNA cassette induces humoral and cell-mediated immune responses and protects immunized mice against a lethal influenza virus challenge. Thus, the LNP-encapsulated linear expression cassette may provide a safer approach for rapid responses to infectious diseases in animals.