Mucosal Adjuvant Activity of a PAR-2–activating Peptide Enhances Lymph Node Immune Cell Recruitment and Promotes Immune Cell Activation in a Prime–boost Influenza Vaccination Model

Influenza A virus (IAV) remains a global health challenge, and current intramuscular vaccines often fail to induce strong mucosal immunity in the airways. We have developed a novel intranasal vaccination strategy in mice using a Protease-Activated Receptor 2 (PAR-2)-activating peptide (AP) as an adjuvant co-delivered with influenza virosomes. We have previously shown that this peptide enhances virosome-induced CD8+ T cell responses and protection against lethal IAV challenge. Here we studied the immune changes induced by this peptide in draining lymph nodes and the lung. We show that AP promotes accumulation of lymphoid and myeloid cells to the mediastinal lymph nodes, including CD3+ T cells and CD11b-/CD11b+ dendritic cells (DCs), and enhances DC maturation and CCR7 expression, which facilitates migration to lymph nodes. Along with accumulation of antigen-presenting cells, we detected increases in naïve, central memory (TCM), and effector memory (TEM) T cells. In addition, AP promoted the presence of tissue-resident memory T cells (TRM) and monocytes in the lungs, supporting rapid tissue-targeted immunity. These results demonstrate that AP functions as a potent mucosal adjuvant by enhancing early immune cell recruitment, DC activation, and adaptive T cell responses in lymph nodes, ultimately promoting tissue-resident immunity. Our findings highlight the potential of PAR-2 activation to improve efficacy of mucosal influenza vaccines.