Unmethylated CpG motifs are capable of evoking a range of immunostimulatory effects in vertebrates and have tremendous potential to be used as therapeutic agents and adjuvants. This particular type of CpG motif has been demonstrated to be an excellent immune adjuvant mediated by Toll-like receptor 9 (TLR9) in various mammalian vaccines; however, only a few studies confirm its efficacy in avian vaccines. In the present study, immunomodulatory activities of plasmids with various copy numbers of a CpG motif were evaluated in chickens inoculated with an avian influenza vaccine. Results showed that the plasmid with 64 copies of the CpG motif (64CpG-plasmid) significantly enhanced the mRNA expressions of interferon-gamma (IFN-γ), TLR3 and TLR7 in chicken splenocytes compared to plasmids with lesser copies of the CpG motif in vitro. Chickens inoculated with the H5N2 avian influenza inactivated vaccines (V52) coadministrated with the 64CpG-plasmid (V52-64CpG) showed significant increments of hemagglutination inhibition (HI) titers, peripheral blood mononuclear cell (PBMC) proliferation, and mRNA expressions of IFN-α, IFN-γ, TLR3, TLR7 and TLR21 in splenocytes as compared to those of chickens inoculated with V52 alone, V52 adjuvanted with aluminum gel (V52-gel), or with V52-gel plus vector. Additionally, following challenge with a highly virulent H5N1 strain, a higher survival rate (100%) was observed in chickens inoculated with V52-64CpG as compared to those that received V52-gel (80%) or PBS (0%). The 64CpG-plasmid significantly enhanced chicken immunity in vitro and in vivo; thus it can be a potent adjuvant in an avian influenza vaccine for chickens.
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