DNA vaccines use plasmid DNA to induce protective immunity against influenza by various routes, including parenteral, mucosal, and gene-gun delivery. In trials, DNA expressing influenza hemagglutinin glycoproteins protected 67–95% of mice and 25–63% of chickens against lethal influenza challenges. Parenteral routes like intramuscular and intravenous injections provided good protection, while mucosal routes such as intranasal and intratracheal administration also showed effectiveness. The most efficient method was gene-gun delivery, which achieved 95% protection with as little as 0.4 μg of DNA. This method is highly effective due to its ability to deliver DNA to the epidermis, where it is efficiently expressed and presented to the immune system. DNA vaccines represent a new approach to subunit vaccine development, offering the potential for highly effective immunization with minimal DNA. The study evaluated the effectiveness of various DNA inoculation routes, finding that the efficiency of transfection does not necessarily correlate with vaccination efficiency. Intramuscular and intravenous routes provided good protection, while mucosal routes also showed effectiveness. Gene-gun delivery was the most efficient, requiring significantly less DNA than saline injections. The study also demonstrated that DNA vaccines can induce antibody responses, with IgG levels increasing after challenge. Memory responses were observed, with vaccinated animals showing secondary antibody responses. The results indicate that DNA vaccines can be effective through multiple routes, with gene-gun delivery and mucosal administration being particularly promising. These findings highlight the potential of DNA vaccines for developing subunit vaccines with high efficacy and minimal DNA requirements.DNA vaccines use plasmid DNA to induce protective immunity against influenza by various routes, including parenteral, mucosal, and gene-gun delivery. In trials, DNA expressing influenza hemagglutinin glycoproteins protected 67–95% of mice and 25–63% of chickens against lethal influenza challenges. Parenteral routes like intramuscular and intravenous injections provided good protection, while mucosal routes such as intranasal and intratracheal administration also showed effectiveness. The most efficient method was gene-gun delivery, which achieved 95% protection with as little as 0.4 μg of DNA. This method is highly effective due to its ability to deliver DNA to the epidermis, where it is efficiently expressed and presented to the immune system. DNA vaccines represent a new approach to subunit vaccine development, offering the potential for highly effective immunization with minimal DNA. The study evaluated the effectiveness of various DNA inoculation routes, finding that the efficiency of transfection does not necessarily correlate with vaccination efficiency. Intramuscular and intravenous routes provided good protection, while mucosal routes also showed effectiveness. Gene-gun delivery was the most efficient, requiring significantly less DNA than saline injections. The study also demonstrated that DNA vaccines can induce antibody responses, with IgG levels increasing after challenge. Memory responses were observed, with vaccinated animals showing secondary antibody responses. The results indicate that DNA vaccines can be effective through multiple routes, with gene-gun delivery and mucosal administration being particularly promising. These findings highlight the potential of DNA vaccines for developing subunit vaccines with high efficacy and minimal DNA requirements.