The Enhancement Of Novel CpG ODN On The Efficacy Of Hepatitis B Vaccine

Hepatitis B virus (HBV) causes acute and chronic infection in human and is responsible for substantial morbidity and mortality. Up to 2004, more than 400 million people have infected with HBV worldwide. Vaccination with HBsAg vaccines consisting of recombinant hepatitis B surface antigen (HBsAg) adsorbed to aluminum adjuvant is albe to stimulate protective antibodies against HBV in majority of people. However, there are still 10% of people who are failed to produce protective antibodies after the vaccination with HBsAg vaccines. So it is very important to develop a more efficient HBsAg vaccine that can stimulate the 10% of people to produce high titer protective antibodies against HBV. In recent years, oligodeoxynucleotides (ODN) containing unmethylated CpG motifs have been shown to enhance humoral immune response as molecular adjuvants for some vaccines. In the process of large scale of screening and identifying of self-designed CpG ODNs, we have found a novel CpG ODN designated as CpG-684 with B type CpG ODN activities which made it a ideal adjuvant candidate for developing a CpG ODN enhanced HBsAg vaccine. The CpG-684 is demonstrated to have the following activities: 1.CpG-684 can activate human PBMC in vitro To test the stimulating activity of CpG-684, we first observed whether CpG-684 could stimulat the proliferation of human PBMC using 3H-TdR incorrpertion. The result showed that CpG-684 stimulated human PBMC to proliferate. Next we obseved activiting effect of CpG-684 on human PBMC using two color double antibody fluressent staining. The result demonstrated that CpG-684 upregulated CD69 expression in CD19+, CD3+and CD56+ positive cells in human PBMC, indicating CpG-684 could activate human B cells, T cells and NK cells. We also found that CpG-684 could upregulate the expression of HLA-A2 in human PBMC, which indicated CpG-684 could enhance the efficiency of APC presenting the HLA-1 restricted antigen. Together, these results revealed that CpG-684 could be used as molecular adjuvant of hepatitis B vaccine. 2. CpG-684 can activate murine lymphocyte in vitro To test the stimulating activity of CpG-684, we first observed whether CpG-684 could stimulat the proliferation of murine lymphocyte using 3H-TdR incorrpertion. The result showed that CpG-684 stimulated murine lymphocyte to proliferate. Next we observed murine lymphocyte avtiviation caused by CpG-684 using two color double antibody fluressent staining. The result demonstrated that CpG-684 could upregulated CD69 expression in CD19+, CD4+and CD8+ positive cells in murine lymphocyte, indicating CpG-684 could activate mouse B cells, T cells. We also found that CpG-684 could upregulate the expression of MHC-Ⅰin mouse lymphocyte, which indicated CpG-684 can enhance the efficiency of APC presenting the MHC-Ⅰrestricted antigen. 3.CpG-684 enhances the efficacy of hepatitis B vaccine in BALB/c immunized by i.m BALB/c mice were peritoneally injected with HBsAg, HBsAg/alum,HBsAg/CpG-684 or HBsAg/alum/CpG respectively on day 0 and 42. On day 14 after the first immuniziation and on day 7 after second immunization, the sera of immunized mice were collected for testing the level of Ab against HBsAg (anti-HBs) by ELISA. The result showed that the level of anti-HBsAg antibody in mice immumized with HBsAg/alum/CpG is 1.5 times higher than those of mice immunized with HBsAg/alum on day 14 after first immunization. On day 7 after second immunization, the level of anti-HBsAg antibody in mice immumized with HBsAg/alum/CpG is 6 times higher than those of mice immunized with HBsAg/alum. These results indicated that CpG-684 is potent adjuvant to hepatitis B vaccine. 4. CpG-684 at different doses enhances the immunogenicity of hepatitis B vaccine in BALB/c immunized by i.m According to the results that CpG-684 can enhance the immunogenicity of HBsAg vaccines in BALB/c, we designed a experiment to determin the optimal dose of CpG-684. BALB/c mice were immunized on day o and 28 with HBsAg vaccine plus CpG-684 at 1.5625μg,6.25μg,12.5μg,50μg,100μg,500μg. On day 48 and 84 after second immunization the sera of immunized mice were colleceted for testing the level of anti-HBsAg antibody by ELISA. The result showed that the optimal dose of CpG-684 is 12.5μg. 5. CpG-684 enhances the efficacy of hepatitis B vaccine in BALB/c immunized by i.p BALB/c mice were intraperitoneally injection with HBsAg, HBsAg/alum, HBsAg/CpG-684 or HBsAg/alum/CpG684 on day 0. On day 28 and 63 after...