In 2009, a novel swine-origin H1N1 virus emerged, causing a pandemic. The virus, known as H1N1pdm09, quickly displaced the circulating H1 lineage and became the dominant seasonal influenza A virus subtype infecting humans. Human-to-swine spillovers of the H1N1pdm09 have occurred frequently, and each occurrence has led to sustained transmission of the human-origin H1N1pdm09 within swine populations. In the present study, we developed a lipid nanoparticle-based DNA vaccine (LNP-DNA) containing the hemagglutinin gene of a swine-origin H1N1pdm09. In pigs, this LNP-DNA vaccine induced a robust antibody response after a single intramuscular immunization and protected the pigs against challenge infection with the homologous swine-origin H1N1pdm09 virus. In a mouse model, the LNP-DNA vaccine induced antibody and T-cell responses and protected mice against lethal challenge with a mouse-adapted human-origin H1N1pdm09 virus. These findings demonstrate the potential of the LNP-DNA vaccine to protect against both swine- and human-origin H1N1pdm09 viruses. IMPORTANCE: Swine influenza A virus (IAV) is widespread and causes significant economic losses to the swine industry. Moreover, bidirectional transmission of IAV between swine and humans commonly occurs. Once introduced into the swine population, human-origin IAV often reassorts with endemic swine IAV, resulting in reassortant viruses. Thus, it is imperative to develop a vaccine that is not only effective against IAV strains endemic in swine but also capable of preventing the spillover of human-origin IAV. In this study, we developed a lipid nanoparticle-encapsulated DNA plasmid vaccine (LNP-DNA) that demonstrates efficacy against both swine- and human-origin H1N1 viruses. The LNP-DNA vaccines are non-infectious and non-viable, meeting the criteria to serve as a vaccine platform for rapidly updating vaccines. Collectively, this LNP-DNA vaccine approach holds great potential for alleviating the impact of IAV on the swine industry and preventing the emergence of reassortant IAV strains.

Maternally derived antibodies (MDAs) play a vital role in protecting neonates from infectious diseases, but their presence at the time of vaccination can interfere with vaccine-induced immune responses, thereby reducing vaccine effectiveness. MDA interference has been documented in pigs vaccinated with whole-inactivated virus (WIV) vaccines against swine influenza A virus (IAV). This study evaluated the efficacy of a lipid nanoparticle-encapsulated DNA (LNP-DNA) vaccine against swine IAV in the presence and absence of MDAs, comparing it to a WIV vaccine. In MDA-negative piglets, both the LNP-DNA and WIV vaccines induced strong immune responses and effectively prevented the vaccinated animals from being infected with the homologous IAV strain. However, in MDA-positive piglets, the WIV vaccine failed to trigger significant antibody or T-cell responses and offered no protection against viral shedding or lung damage. In contrast, the LNP-DNA vaccine elicited stronger immune responses in MDA-positive pigs, reduced nasal viral shedding, and prevented lung lesions. These findings demonstrate that the LNP-DNA vaccine overcomes MDA interference, making it a promising strategy for enhancing vaccine efficacy in neonatal animals with maternal antibodies. IMPORTANCE: Maternally derived antibody (MDA) interference is a major obstacle to developing effective vaccines for neonates. In pigs, MDAs significantly impair immune responses to a whole-inactivated virus vaccine. Here, we show that vaccination with a lipid nanoparticle (LNP)-encapsulated DNA vaccine can partially overcome MDA interference. These findings underscore the potential of the LNP-DNA vaccine as a viable strategy for effectively immunizing MDA-positive populations. Additionally, LNP-DNA vaccination in young pigs provides a valuable model for exploring the immunological mechanisms behind MDA-mediated suppression of vaccine-induced immunity.

To investigate the role of PRRSV nonstructural proteins (nsps) in viral RNA replication and transcription, we generated a cDNA clone of PRRSV strain NCV1 carrying the nanoluciferase (nluc) gene under the control of the transcription regulatory sequence 6 (TRS6) designated as pNCV1-Nluc. Cells transfected with the pNCV1-Nluc DNA plasmid produced an infectious virus and high levels of luciferase activity. Interestingly, cells transfected with mutant pNCV1-Nluc constructs carrying deletions in nsp7 or nsp9 regions also exhibited luciferase activity, although no infectious virus was produced. Further investigation revealed that the cDNA sequences corresponding to the PRRSV 5′ untranslated region (UTR) and TRS, when cloned upstream of the reporter gene nluc, were able to drive the expression of the reporter genes in the transfected cells. Luciferase signals from cells transfected with a reporter plasmid carrying PRRSV 5′ UTR or TRS sequences upstream of nluc were in the range of 6- to 10-fold higher compared to cells transfected with an empty plasmid carrying nluc only. The results suggest that PRRSV 5′ UTR and TRS-B in their cDNA forms possess cryptic eukaryotic promoter activity.

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. IMPORTANCE: Lipid 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.