The Study Of Immune Enhancement Efficacy Of CpG Adjuvant On The Eg95 Antigen Of Echinococus Granulosus

Bacterial genomic DNA contains CpG motifs, in which an unmethylated cytosine-phosphate-guanosine(CpG) dinucleotide is flanked by two 5’ purines and two 3’ pyrimidines CpG ODN is an oligodeoxynucleotide containing multiple CpG motifs. CpG ODN can activate pDCs, B lymphocytes, monocytes and other immune cells, hence activate the innate immune and promote the secretion of IL12, TNF-α, IFN-α / β and IFN-γ and other cytokines CpG ODN stimulates an cytokine profile that is in favor of Th1 type immune response by engaging the TLR9 receptor of immune cells. The optimal CpG ODN sequence for activating mouse immune cells is GACGTT, so it can be used as a potential immune enhancer. Plasmid pUC18-CpG is constructed by cloning 10 copies of GACGTT into the pUC18 vector.RAW264.7 cells were stimulated by pUC18-CpG and ODN, respectively. Total RNA was extracted and reverse transcribed into cDNA. The quantification of cytokine expression was determined by real-time PCR using the cDNA as template. The results showed that after stimulation by CpG ODN and pUC18-CpG, the relative expression levels of IFN-β, IL-1β, TNF-α and IL-12p40 of pUC18-CpG group and the CpG ODN group were 11.17±1.34, 10.61±0.69, 5.56±0.41, 51.17±2.56和17.22±1.33, 64.56±3.87, 4.45±0.25, 9.50±4.87 respectively. Both CpG groups showed higher cytokine expression than that of the control group(P<0.05) except for IFN-γ. The relative cytokine expression levels from CpG ODN Group is more significant than that of the pUC18-CpG Group except for TNF-α(P<0.05).Escherichia coli containing pET-32a-3Eg95 was induced by IPTG, and then the recombinant 3Eg95 protein was expressed and later harvested by affinity chromatography. The obtained antigen was mixed with the conventional adjuvant Quli-A, pUC18-CpG and CpG ODN, respectively, to prepare vaccine candidates for mice immunization. After vaccination, the average levels of serum IgG antibody were tested by indirect ELISA at different time points and the quantification of cytokine expression were determined by real-time PCR after in vitro stimulation. Results showed that the average levels of serum IgG antibody(OD450nm) of the pUC18-CpG group and the CpG ODN group on Day 14,28 and 42 were 3.10±0.07, 3.03±0.15, 3.22±0.05 and 2.98±0.10, 3.12±0.06, 3.27±0.33,respectively. Antibody levels from both CpG groups were significantly higher compared to the conventional adjuvant Quli-A group(P<0.05). The average level of serum IgG antibody in CpG ODN group was a little higher than that in pUC18-CpG group(P≥0.05). After Stimulation by recombinant protein 3Eg95, the relative expression levels of IFN-β、IFN-γ and TNF-α of pUC18-CpG Group and the CpG ODN Group were 6.88±0.32, 2.35±0.45, 6.28±0.21 and 5.03±0.50, 2.85±0.18, 7.07±1.01, respectively, and both were higher than that of the Quli-A Group(P<0.05).Therefore, pUC18-CpG and CpG ODN can increase the expression of type I IFN and proinflammatory cytokines during in vitro stimulation, with the characteristics of inducing a Th1 type immune response. The pUC18-CpG and CpG ODN have significant immune enhancement property on the 3Eg95 antigen in both humoral and cellular immune response, while the immune enhancement activity of CpG ODN is slightly better than that of pUC18-CpG. In conclusion, both pUC18-CpG and CpG ODN could be used as efficient immune adjuvants in future vaccines against Echinococcus granulosus infection.