Influenza whole inactivated virus (WIV) is more immunogenic and induces protective antibody responses compared with otherformulations, like split virus or subunit vaccines, after intranasal mucosal delivery. Polyethyleneimine (PEI), an organic polycation, is widely used as a reagent for gene transfection and DNA vaccine delivery. Although PEI recently has demonstrated potent mucosal adjuvant activity for viral subunit glycoprotein antigens, its immune activity with H9N2 WIV is not well demonstrated. Here, mice were immunized intranasally with H9N2 WIV combined with PEI, and the levels of local respiratory tract and systemic immune responses were measured. Compared to H9N2 WIV alone, antigen-specific IgA levels in the local nasal cavity, trachea, and lung, as well as levels of IgG and its subtypes (IgG1 and IgG2a) in the serum, were strongly enhanced with the combination. Similarly, the activation and proliferation of splenocytes were markedly increased. In addition, PEI is superior as an H9N2 WIV delivery system due to its ability to greatly increase the viral adhesion to mucosal epithelial cells and to enhance the cellular uptake and endosomal escape of antigens in dendritic cells (DCs) and further significantly activate DCs to mature. Taken together, these results provided more insights that PEI has potential as an adjuvant for H9N2 particle antigen intranasal vaccination.T he rise and spread of the low-pathogenic avian H9N2 influenza virus have seriously increased the risk of a new influenza pandemic. H9N2 viruses have prevailed in chickens in China in recent years and have constantly undergone reassortment, and novel genotypes have continued to emerge (1-3). A novel H7N9 reassortant subtype was recently found to cause severe human respiratory infections in China (4). Bioinformatic analyses for the H7N9 virus revealed that its six internal genes were from H9N2 avian influenza viruses of chickens (5). Thus, the elimination of low-pathogenic avian H9N2 influenza virus in poultry becomes even more important in influenza prevention.The nasal cavity of the respiratory tract is the primary entry site of the H9N2 influenza virus, and the viral infection could be discontinued if intranasal immunity is well established (6). Compared with live attenuated influenza vaccines or subunit influenza vaccines (such as purified viral hemagglutinin [HA]) or neuraminidase [NA]) proteins), whole inactivated H9N2 influenza vaccines have more advantages, including an improved safety profile, higher immunogenicity, more effective ability of establishing cross-protection at the pathogen's entry site, and stronger crosspresentation of antigens by dendritic cells (DCs) for a CD8 ϩ T cell response against viruses (7-9). However, mucosal immunization by intranasal delivery with inactivated virus alone is often poorly effective. Unlike systemic immunization, nasal antigens must cross various barriers (compact epithelium, mucociliary clearance, and mucus) before they contact with submucosal immune cells (10). Many researchers used various ...