Avian influenza virus (AIV) causes severe economic losses to poultry industry and has a great potential for becoming humankind pandemic threat in the next future, and vaccination is a common solution to overcome this problem. The proper vaccine administered for rapid intervention, should be efficient and safe. The application of natural adjuvants have opened up new avenues toward reaching highly efficient and secure vaccine in recent years. In this study, we investigated the H9N2 antigen adjuvanted with Mx protein and chitosan, either alone or combined formulation, in BALB/c mouse model. After preparation and inactivation of the H9N2, the characterization of the nanoparticles were evaluated. Then, the H9N2 antigen was encapsulated with chitosan only and in combination with Mx protein. Afterward, the mice were administrated by different ratios of the treatments through intranasal (IN) route. The efficiency and safety of the adjuvant-nanovaccine candidate were evaluated by comparing the antibody level of treated and control groups of animals. The results demonstrated the higher antibody level in treated groups as compared to control samples. Interestingly, we also showed that combination of Mx and chitosan can significantly induce influenza specific antibody titer, indicating to the synergy effects of the applied nanoparticles together. In conclusion, formulation of Mx as an adjuvant and chitosan as a nanocarrier is a promising platform for developing an efficient and safe vaccine against influenza virus.

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