Intranasal and Intramuscular Immunization with a Universal Influenza A Virus Vaccine Induces Distinct and Protective Immune Responses in Mice

Influenza infections cause more than half a million deaths annually. Despite the prevalence of influenza vaccines, the vaccine efficacy varies widely (19-54%) due frequent mutations of the viral genome, a problem exacerbated by cross-species transmission. Although intranasal (IN) administration could enhance effectiveness, most current vaccines are intramuscular (IM) due to limited understanding of the IN mechanism. This study aims to compare the mechanism of protection conferred by an IN or IM delivered universal influenza vaccine. We designed a vector expressing a conserved antigen, the nucleoprotein (NP), fused with CD40 ligand, acting as both a targeting ligand and a molecular adjuvant. Only IN vaccinated mice were protected against H1N1, H3N2, and a highly pathogenic and transmissive H5N1 avian strain, showing high antibody levels and distinct cytokines along the respiratory tract. To further characterize the cell-mediated response, IN or IM vaccinated animals were treated with an agonist for sphingosine-1-phosphate receptors, inhibiting lymphocyte egress. After the treatment, only IN was protective against the viral challenge, indicating that IN induced a potent local cell-mediated defense without circulating lymphocytes. Further flow cytometric analysis revealed that IN induced proliferative tissue-resident T cells in the lungs. These findings suggest that IN vaccination induces superior cross-subtype protection by stimulating robust mucosal immunity.