Improved cellular immune response induced by intranasal boost immunization with chitosan coated DNA vaccine against H9N2 influenza virus challenge.

The H9N2 avian influenza virus (AIV) is spreading worldwide. Presence of H9N2 virus tends to increase the chances of infection with other pathogens which can lead to more serious economic losses. In a previous study, a regulated delayed lysis Salmonella vector was used to deliver a DNA vaccine named pYL233 encoding M1 protein, mosaic HA protein and chicken GM-CSF adjuvant. To further increase its efficiency, chitosan as a natural adjuvant was applied in this study. The purified plasmid pYL233 was coated with chitosan to form a DNA containing nanoparticles (named CS233) by ionic gel method and immunized by intranasal boost immunization in birds primed by oral administration with Salmonella strain. The CS233 DNA nanoparticle has a particle size of about 150 nm, with an encapsulation efficiency of 93.2 ± 0.12 % which protected the DNA plasmid from DNase I digestion and could be stable for a period of time at 37°. After intranasal boost immunization, the CS233 immunized chickens elicited higher antibody response, elevated CD4 T cells and CD8 T cells activation and increased T-lymphocyte proliferation, as well as increased productions of IL-4 and IFN-γ. After challenge, chickens immunized with CS233 resulted in the lowest levels of pulmonary virus titer and viral shedding as compared to the other challenge groups. The results showed that the combination of intranasal immunization with chitosan-coated DNA vaccine and oral immunization with regulatory delayed lytic Salmonella strain could enhance the immune response and able to provide protection against H9N2 challenge.