Tehran University of Medical Sciences Iranian Journal of Microbiology 2008-3289 18 5 2026 10 05 841 860 Khabat Barkhordari Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Farhad Rezaei Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Vahid Salimi Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Kaveh Sadeghi Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Mostafa Alavi Moghadam Department of Emergency Medicine, Imam Hossein Hospital, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Hossein Bagheri Department of Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran Masoumeh Pourjabali Department of Pathology, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran Maryam Karimi Department of Biostatistics, School of Allied Medical Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran Omid Fotouhi Science for Life Laboratory, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Solna, Sweden Talat Mokhtari Azad Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2026 07 28 2026 07 28 Background and Objectives: Influenza A virus remains a major global health concern because antigenic drift reduces the long-term efficacy of current vaccines. This study evaluated a multi-epitope DNA vaccine targeting conserved regions of hemagglutinin (HA) and polymerase acidic (PA) proteins from influenza A/H1N1 and A/H3N2 viruses. Materials and Methods: Conserved B-cell, CD4⁺ T-cell, and CD8⁺ T-cell epitopes were identified using immunoinformatic tools and linked to β-defensin-2 in the pcDNA3.1(+) vector. Protein expression was confirmed in HEK293 cells. BALB/c mice were immunized intramuscularly and challenged with lethal doses of H1N1 or H3N2 viruses. Humoral and cellular immune responses, lung viral loads, histopathology, and clinical outcomes were assessed. Results: Vaccinated mice exhibited undetectable or markedly reduced lung viral loads (1,000-10,000-fold; p<0.01), significantly decreased pulmonary lesions, and milder clinical manifestations than controls. Vaccination induced significant CD4⁺ (p=0.041) and CD8⁺ (p=0.038) T-cell responses in bronchoalveolar lavage fluid, accompanied by increased interferon-γ and interleukin-4 production, indicating balanced cellular and humoral immunity. Complete protection against mortality was observed following viral challenge. Conclusion: This multi-epitope DNA vaccine elicited cross-protective immunity against influenza A/H1N1 and A/H3N2 by targeting conserved HA and PA epitopes, supporting its potential as a candidate for universal influenza vaccine development. https://ijm.tums.ac.ir/index.php/ijm/article/view/6355 https://ijm.tums.ac.ir/index.php/ijm/article/download/6355/1944