Effects of Antigen Dosage and Chitosan Micro/Nanoparticle Size on Immune Responses in Mice Immunized with H5N1 Influenza Vaccine.

Highly pathogenic avian influenza A/H5N1 remains a persistent threat to public health and poultry production. H5N1 antigens are typically poorly immunogenic and require effective adjuvants for antigen dose-sparing. Here, we evaluated chitosan microparticles (CSMs) and nanoparticles (CSNs) as polymeric nano-adjuvants for an H5N1 influenza vaccine, focusing on the roles of antigen dose and particle size. A purified hemagglutinin antigen was adsorbed onto chitosan particles at doses ranging from 0.15 to 5.0 µg. Both CSNs and CSMs showed consistently high loading efficiency (97-99%). BALB/c mice were immunized intramuscularly in a prime-boost schedule. Chitosan nanoparticles significantly enhanced IgG and hemagglutination inhibition (HI) titers at low antigen doses compared with aluminum hydroxide and antigen-only controls (p < 0.05). Immune responses reached saturation at a 1.5 µg dose of antigen for chitosan nanoparticles and 3.0 µg for chitosan microparticles. IgG subtype analysis suggested a balanced IgG1/IgG2a profile. Collectively, these findings support chitosan-based polymeric nanoparticles as promising adjuvants enabling dose-sparing H5N1 vaccination.