Genetically engineered M13 phage-mediated H9N2 DNA vaccine with enhanced mucosal and systemic immune responses in mice

Vaccine adjuvants and delivery systems have long been used in DNA vaccines to enhance immunogenicity. In this study, we developed a DNA vaccine delivery platform by combining <i>N</i>-2-hydroxypropyl trimethyl ammonium chloride chitosan/carboxymethyl chitosan nanoparticles (N-2-HACC/CMCS NPs) with a genetically engineered M13 phage containing the HA gene of H9N2 AIV (HA-M13). The composite NPs (HA-M13/N-2-HACC/CMCS) had an average particle size of 135.24 ± 4.36 nm, and the HA gene encapsulated in the composite NPs could be expressed <i>in vitro</i>. Additionally, the N-2-HACC/CMCS NPs exhibited high stability and effectively protected the HA gene and M13 phage from degradation while sustaining antigen release. Furthermore, the N-2-HACC/CMCS NPs promoted the maturation of DC2.4, enhanced MHC I and MHC II pathways and improved cellular, humoral, and mucosal immune responses. Mice immunized with HA-M13/N-2-HACC/CMCS via nasal and intramuscular injections presented higher anti-H9N2 AIV antibody titers than those given the commercial vaccine. Lymphocyte proliferation, as well as the levels of the cytokines IL-2, IL-4, IFN-<i>γ</i>, CD4⁺, and CD8⁺ T lymphocyte levels, also significantly increased. The nanovaccine provided effective protection against H9N2 AIV infection for 154 days postimmunization, surpassing the 120-day protection provided by the commercial vaccine. Consequently, the N-2-HACC/CMCS NPs loaded with M13 phages exhibit significant potential as vaccine adjuvants and mucosal immune delivery system.