The study evaluates the effectiveness of various routes of DNA inoculation in inducing protective immunity against influenza. Plasmid DNAs expressing influenza virus hemagglutinin glycoproteins were tested in mice and chickens. Intramuscular and intravenous injections provided good protection, while mucosal routes such as intranasal and intratracheal inoculations also showed efficacy. The most efficient method was gene gun delivery, which required significantly less DNA (0.4 μg) to achieve 95% protection in mice. The study highlights the potential of DNA vaccines for subunit vaccines, emphasizing the importance of efficient antigen presentation and recognition in addition to transfection efficiency.The study evaluates the effectiveness of various routes of DNA inoculation in inducing protective immunity against influenza. Plasmid DNAs expressing influenza virus hemagglutinin glycoproteins were tested in mice and chickens. Intramuscular and intravenous injections provided good protection, while mucosal routes such as intranasal and intratracheal inoculations also showed efficacy. The most efficient method was gene gun delivery, which required significantly less DNA (0.4 μg) to achieve 95% protection in mice. The study highlights the potential of DNA vaccines for subunit vaccines, emphasizing the importance of efficient antigen presentation and recognition in addition to transfection efficiency.