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PLoS One. 2014 Sep 30;9(9):e108808. doi: 10.1371/journal.pone.0108808. eCollection 2014.

Development of CpG-oligodeoxynucleotides for effective activation of rabbit TLR9 mediated immune responses.

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Immunology Research Center, National Health Research Institutes, Miaoli, Taiwan; Program in Environmental and Occupational Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
Immunology Research Center, National Health Research Institutes, Miaoli, Taiwan.
Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan.
Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan.
Institute of Molecular Medicine, National Taiwan University, Taipei, Taiwan.


CpG-oligodeoxynucleotides (CpG-ODN) are potent immune stimuli being developed for use as adjuvants in different species. Toll-like receptor 9 (TLR9) is the cellular receptor for CpG-ODN in mammalian cells. The CpG-ODN with 18-24 deoxynucleotides that are in current use for human and mouse cells, however, have low activity with rabbit TLR9. Using a cell-based activation assay, we developed a type of CpG-ODN containing a GACGTT or AACGTT motif in 12 phosphorothioate-modified deoxynucleotides with potent stimulatory activity for rabbit TLR9. The developed CpG-ODN have higher activities than other developed CpG-ODN in eliciting antigen-nonspecific immune responses in rabbit splenocytes. When mixed with an NJ85 peptide derived from rabbit hemorrhagic disease virus, they had potent activities to boost an antigen-specific T cell activation and antibody production in rabbits. Compared to Freund's adjuvant, the developed CpG-ODN are capable of boosting a potent and less toxic antibody response. The results of this study suggest that both the choice of CpG-motif and its length are important factors for CpG-ODN to effectively activate rabbit TLR9 mediated immune responses.

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