Effects of different CpG oligodeoxynucleotides with inactivated avian H5N1 influenza virus on mucosal immunity of chickens.

Oligodeoxynucleotide containing unmethylated CpG motifs (CpG-ODN) has been proved to be a potent and safe vaccine adjuvant. However, the application of CpG-ODN in poultry vaccines was limited because of its high cost to benefit ratio. The objective of this study was to identify the CpG-ODN with efficient adjuvant activity and low cost in chickens. Four sequences of CpG-ODN were designed based on CpG-ODN 2006, which was used as a template and positive sequence in our study. In the current study, in vitro observations revealed that the designed CpG-ODN had efficient immunostimulatory effects on chicken splenic lymphocytes. The in vivo results showed that the mRNA expressions of IL-6, IL-12, interferon-γ, and Toll-like receptor (TLR) 21 in upper respiratory tract tissues increased significantly in the early period after intranasal immunization with inactivated avian H5N1 influenza virus (IAIV) and CpG-ODN (P < 0.01). In addition, the avian influenza virus (AIV)-specific secretory IgA antibody level in the lavage fluid of upper respiratory tract increased significantly after intranasal immunization with IAIV and CpG-ODN, so did AIV-specific IgG in serum (P < 0.01). Among all the designed CpG-ODN, CpG-ODN F3 with an addition of poly-guanosine strings at the 3'-end not only had the best enhancement on local mucosal immune response but also showed an effective induction of systemic immune response. Most importantly, the virus challenge study showed that prior administration of IAIV with CpG-ODN F3 could protect chickens effectively against live AIV H5N1 challenge. Additionally, among all the CpG-ODN in our study, the cost of the designed CpG-ODN F3 was the lowest because of the partially phosphorothioate backbone. Therefore, we speculated that CpG-ODN F3 with efficient adjuvant activity and a big cost advantage over CpG-ODN F1 (CpG-ODN 2006) might serve as an efficient and affordable nasal adjuvant for inactivated AIV vaccine in chicken.