Objective: Flu is a devastating disease in poultry industry. However, there is not an effective vaccine against it, because the major virus antigen (hemagglutinin) undergoes many changes during circulation in the field. Therefore, it is of great importance to develop vaccines against conserved structures of the virus. The other challenge is delivery of the vaccine to the major players of immune system such as antigen presenting cells (APC). In the present work, we investigated the effect of a DNA vaccine based on the external domain of Matrix protein (M2e) and nucleoprotein (NP) gene, which was loaded into E.coli Top10 and O78 K80 bacterial ghosts on the expression profile of pro-inflammatory cytokines (PIC) in chicken macrophage primary cells. Materials & Methods: The antigenic cassette containing M2e and NP was synthesized by GenScript Company (USA), and sub-cloned into vector pIRES2-EGFP and named pIRES-Ag E.coli Top10 and O78K80 ghosts were prepared by controlled expression of E-lysis gene. The ghosts were loaded with pIRES-Ag vector by re-suspending the ghosts in a solution containing high concentration of pIRES2-Ag. Subsequently, the already Ficoll extracted monocytes were challenged by the loaded and unloaded ghosts. Succession of the monocytes\' transfection by the ghosts was assessed by fluorescens microscopy and western blot analysis. Afterward, RNA was extracted, and cDNA was synthesized. Relative qPCR experiment was performed for eight PICs on the samples, and finally statistical calculations were done on the obtained results. Results & Conclusion: The efficacy of monocyte transfection by the ghosts was as high as near 90%. The antigenic segment of the construct also finely expressed as corresponding bond appeared in the blot. Moreover, the qPCR results demonstrated the loaded ghosts had a greater impact on the PIC expression for IL1β, IL6, IL8, IL10, IL18 and TNFα. In addition, the loaded or unloaded O78 K80 ghost had a better impact on the PIC induction in compare to Top10 strain. However, iNOS expression was insensitive to the bacterial strain. Surprisingly, induction of LITAF in response to the ghosts was much lower than the same strain that inactivated by boiling, which demonstrate the safety of this method. Regarding to the excellent efficiency of transfection, strong PIC induction and also its safety, this method could have a promising perspective. In the future, this work will be done in an in vivo model.

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