Study On The Immunostimulatory Activity Of A New Efficient Adjuvant CpG Oligodeoxynucleotides On Porcine Immune Cells

CpG motif is a 6-base DNA motif consisting of an unmethylated CpG dinucleotide flanked by two 5'purines and two 3'pyrimidines. Synthetic Oligodeoxynucleotides (ODN) containing CpG motif were demonstrated to mimic a danger signal of bacterial or viral DNA and induce a potent Th1 biased immune response. Therefore, CpG ODN may have potential as a new adjuvant. Further studies documented that different motifs are optimally stimulatory in different species. The current work examines the structure character of the ODN which can stimulate porcine immune cells in vitro and provides an experimental foundation for intensive study on vaccine adjuvant for swine.Using lymphocyte proliferative response (LPR) and IFN- Y secretion assays, a panel of 38 ODN was screened for immunostimulatory activity on porcine peripheral blood mononuclear cells (PBMC) . The PBMC were cultured with ODN and the stimulation index was determined by 3H-TdR incorporation assay. Results show that the immunostimulatory CpG ODN has the following structure: (1)The CpG dinucleotide is needed; (2)The oligonucleotide is optimal which contains 3 or 4 motifs and whose length is between 20-25bp; (3)The modification of the backbone of the CpG ODN has a prominent influence on its immunostimulatory activity. The amount of IFN-γ in the culture supernatant was measured by swIFN-γ kit which is a solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA) . This work demonstrates that the ODN which can stimulate porcine PBMC to secrete IFN- γ has the following structure : It has mixed phosphodiester/phosphorothioate backbone and contains a single hexameric purine/pyrimidine/CG/purine/pyrimidine motif flanked by self-complementary bases that form a stem-loop structure capped at the 3' and 5' end by a polyG tail. These findings indicate that porcine PBMC recognize and respond to two structurally distinct classes of CpG ODN. One class preferentially induces cell proliferation, whereas the other stimulates IFN- γ release. The distinct immunostimulatory properties of the two classes of CpG ODN improve the design of CpG ODN as vaccine adjuvants.