Vaccination through mucosal surfaces has been shown to elicit antiviral immune responses against a number of mucosal pathogens. Here we demonstrate that both mucosal and systemic immune responses can be elicited against a model HIV-1 CN54gp140 antigen when cation-complexed plasmid DNA vaccines are applied topically to the murine pulmonary mucosa as an immune priming strategy. Furthermore, using an influenza challenge model we show that a plasmid DNA vaccine complexed to a less toxic form of PEI called dPEI (a nearly fully hydrolysed linear PEI with 11% additional free protonatable nitrogen atoms) can provide significant protection against a respiratory challenge infection in mice. Furthermore, we show that dPEI polyplexes have the potential to transfect not only mucosal epithelium, but also to enter deeper into tissues through the modulation of tight junction integrity. Taken together, these results demonstrate that less toxic forms of PEI can be effective delivery vehicles for plasmid DNAs to elicit cellular and humoral protective responses in vivo. Moreover, our observations suggest that these less toxic derivatives of PEI could be utilised for topical plasmid DNA vaccine delivery to human mucosal tissue surfaces, and that this application may permit dissemination of the immune responses through the linked mucosal network thus providing protective immunity at distal portals of pathogen entry.