A multi-epitope-based DNA vaccine confers cross-protection against influenza A H1N1 and H3N2 viruses

Abstract

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), signifi- cantly 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.