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Background: In the last decade due to emerge and remerge of influenza viruses, quality improvement of vaccines to increase immune responses in target populations have been more necessary. The potential of biologic adjuvant to stimulate and induce immune system is the basis of modern researches in prevention and controlling program of infectious diseases. In this study, the effect of the coding sequence of cellular Myxovirus resistance (Mx) protein as a biological adjuvant for inducing humoral immune response against influenza virus was investigated. Methods: The experimental study was performed on Balb/c mice in Razi Vaccine and Serum Research Institute from June to November 2014. Three conserved motifs of Mx were selected following sequence alignment between human, mouse and bird species. Potential of the motifs for stimulation immune responses against influenza virus were evaluated using in silico analysis. Based on the immune informatics data Mx1 sequence was the best immune inducer and cloned into pcDNA3.1 vector. Then formulated with inactivated H9N2 influenza antigen as adjuvant and injected to mice groups. The sera of vaccinated mice were collected prior to priming and boosting injections and also at defined weeks and analyzed with serological assays. Histopathological examination was done for evaluation of the vaccine and adjuvant safety. Results: The mean weight of the Balb/c mice in all control and treatment groups was similar and ranged from 21 to 37 gr (P= 0.05). The difference in increasing antibody titers against influenza virus in immunized mice who received Mx1-adjuvanted vaccine especially in second boosting was significant (P= 0.01) compared to the vaccine alone group. More than 78% of the immunized mice receiving two-time boosting have the mean antibody titer of >6 (Log2) which was higher (P= 0.001) comparing to the mice with one booster injection. Conclusion: These data revealed that Mx1 as biological adjuvant was able to increase antibody titer and induction memory immune responses against influenza immunization without causing any side effects. [ABSTRACT FROM AUTHOR]