Stimulatory Effects Of CpG Oligodeoxynucleotides On PBMC From People Infected With Hepatitis B Virus

The unmethylated CpG motif within the bacterial DNA can stimulate the immune system of vertebrates efficiently. Synthetic oligodeoxynucleotides (ODN) that contain CpG motifs are similar to those found in bacterial DNA to stimulate a similar response. At least three structurally distinct types of synthetic CpG ODN have been described: A-, B-and C-type CpG ODN. They can all interact with Toll like receptor 9 (TLR9) to stimulate the immune system. B cells and plasmacytoid dendritic cells (pDCs) are the main human cell types that express TLR9 and respond directly to CpG stimulation. A-type CpG ODN can induce the secretion of IFN-αfrom pDCs, which indirectly supports the subsequent maturationof APCs and activates NK cells. B-type CpG ODN can trigger the proliferation and activation of B cells. C-type CpG ODN has the ability of A-and B-type CpG ODN. The effect of CpG ODN underlies their use as agents against infectious disease, vaccine adjuvants and anti-allergens medicines. Hepatitis B is a kind of infectious diseases that is caused by hepatitis B virus (HBV). According to the investigation of WHO in 2004, about 4 hundred millions people had infected with HBV worldwide and among them 1.2 hundred millions people are Chinese. Among the carriers 30 millions people are chronic hepatitis B patients. Each year, the chronic HBV-related liver diseases cause 350 thousands deaths. The treatment of hepatitis B is a big hard nut in the field of medical science. There are two important antiviral medicines for Hepatitis B: IFN-αand Lamivudine. But both of them have shortcomings. IFN-αcan only be used to the patients with active HBV DNA duplication. However, IFN-αis only effective in 20–40% of patients. With long-term using, it can stimulate the production of antibody to IFN and cause side effects such as anemia. Lamivudine must be used for long-term therapy, which can induce tolerance to the medicine and have no effects to the patients. Because there are so many questions with the medicine used now, it is urgent for us to study new antiviral medicine. CpG ODN is a new kind of immunoregulatory agent. Its therapeutical effects to infectious diseases have many proofs with animal experiments and are being verified by clinical trials. Among the different types of CpG ODN, A-and C-type CpG ODN have the antiviral immunoregulatory effects. During designing and screening CpG ODN, we found a C-type CpG ODN, named as CpG-302, which has obvious antiviral activity; and a B-type CpG ODN, named as CpG-684, that can strongly stimulate the proliferation of B cells. In order to identify if they can be used for Hepatitis B therapy, we tested whether CpG-302 can stimulate PBMC from people infected with HBV to proliferate, produce antiviral factors, enhance the cytotoxicity of NK cell and secrete cytokines. We also compare the difference among the activities of distinct types CpG ODN and analyze the relations between the results and the patients' clinical features using the statistical method. The main results of the study are as follows: 1. Clinical data of people infected with HBV 65 cases of people infected with HBV were included in the study. Among them, 46 are HBV asymptom carriers. The youngest is 16 and the oldest is 74. The average age of these patients is 50.6 and the medium age is 49.5. 20 cases are male and 26 cases are female. 3 cases are positive for HBsAg, HBeAg and HBcAb; 10 cases are positive for HBsAg, HBeAb and HBcAb; 25 cases are positive for HBsAb, HBeAb and HBcAb; 8 cases are positive for HBeAb and HBcAb. 9 are acute hepatitis B patients. The youngest is 19 and the oldest is 46. The average age of these patients is 33.8 and the medium age is 37. 7 cases are male and 2 cases are female. 7 cases are positive for HBsAg, HBeAg and HBcAb; 2 cases are positive for HBsAg, HBeAb and HBcAb. The average value of serum ALT is 293.2u/l. The average value of the quantity of HBV DNA is 5.68×106 copies/ml. 10 are chronic hepatitis B patients. The youngest is 24 and the oldest is 68. The average age of these patients is 41.9 and the medium age is 41.5. 8 cases are male and 2 cases are female. 5 cases are positive for HBsAg, HBeAg and HBcAb; 5 cases are positive for HBsAg, HBeAb and HBcAb. The average value of serum ALT is 283.9u/l. The average value of the quantity of HBV DNA is 1.36×106 copies/ml.In this research, the control group comprises 8 cases and the blood come from the healthy blood donor. 2. Promotion of CpG ODN to the proliferation of PBMC from people infected with HBV The 3H-thymidine incorporation assay was used to test the proliferation of PBMC. The results show: All CpG ODN used in this study can stimulate the proliferation of PBMC from people infected with HBV (p<0.001). The stimulatory effects of CpG-302 are obviously stronger than that of CpG-2216 (the A-type CpG ODN control) (p<0.001). Whereas, such effects of CpG-302 are not significantly different from that of CpG-684, CpG-2006 (the B-type CpG ODN control) and CpG-C274 (the C-type CpG ODN control) (p>0.05). There are no differences in the promotion of CpG-302 to the proliferation of PBMC between people with and without HBV infection (p>0.05). The clinical types of hepatitis B, the serum ALT level, the HBV DNA quantities, the age and gender of patients have no influence on the effects of CpG-302. But such effects of CpG-302 on patients with positive HBsAg, HBeAg and HBcAb are much stronger than that on the patients with positive HBsAb, HBeAb and HBcAb (p<0.05). 3. Stimulation of CpG ODN to PBMC from people infected with HBV to produce antiviral factors The cell protection test was used to examine the ability of CpG ODN to trigger PBMC to produce antiviral factors. The results show: Except for CpG-2006, All CpG ODN tested in this study can stimulate PBMC to produce antiviral factors. The stimulatory function of CpG-302, 2216, CpG-C274 is the most obvious (p<0.001). The stimulatory function of CpG-684 is also strong (p<0.05). The stimulatory function of CpG-302 is obviously stronger than that of CpG-684 and CpG-2006 (p<0.001). Such function of CpG-302 does not have significant difference with that of CpG-2216 and CpG-C274 (p>0.05). The promotion of CpG-302 to the production of antiviral factors by PBMC does not have remarkable differences between people with and without HBV infection (p>0.05). Patients with different clinical manifestations, different virus markers tested by immunological method, different serum ALT level, different HBV DNA quantities, different age and gender have similar response to CpG-302.4. Activation of CpG ODN to NK cells of people infected with HBV The NK cytotoxicity tests were used to examine the activation of CpG ODN to NK cells. The results show: CpG-302 and CpG-2216 can activate NK cells with similar efficiency. 5. Stimulation of CpG ODN to cytokine secretion by PBMC from people infected with HBV The levels of cytokine in supernatant were measured using commercially available ELISA kits. 5.1 Measuring the content of IFN-αin the supernatant The results show: The content of IFN-αin the plasma and supernatant of medium group have no obvious difference (p>0.05). The content of IFN-αin the supernatant of PBMC stimulated by CpG-302 are much higher than that in the plasma (p<0.001). CpG-302 and CpG-2216 can obviously stimulate PBMC to secrete IFN-α(p<0.001). CpG-C274 can also stimulate PBMC to secrete IFN-α(p<0.05). The stimulatory effect of CpG-302 is obviously higher than that of CpG-2006 (p<0.05). Such effects of CpG-302 are not significantly different to that of CpG-2216 and CpG-C274 (p>0.05). 5.2 Measuring the content of IFN-γin the supernatant The results show: The content of IFN-γin the plasma and supernatant of medium group have no obvious difference (p>0.05). The content of IFN-γin the supernatant from PBMC stimulated by CpG-302 are much higher than that in the plasma (p<0.001). CpG-302 can obviously stimulate PBMC to secrete IFN-γ(p<0.001). CpG-2216, CpG-2006, CpG-C274 can also stimulate PBMC to secrete IFN-γ(p<0.01). Such stimulatory effect of CpG-302 does not have significant difference with that of CpG-2216, CpG-2006 and CpG-C274 (p>0.05). 5.3 Measuring the content of IL-12 in the supernatant The results show: The content of IL-12 in the supernatant of the medium group are similar to that in the plasma (p>0.05). The content of IL-12 in the supernatant from PBMC stimulated by CpG-302 are similar to that in the plasma (p>0.05). The stimulatory activity of CpG-302, CpG-2216, CpG-2006 and CpG-C274 is similar (p>0.05). 5.4 Measuring the content of IL-6 in the supernatant The results show: The content of IL-6 in the supernatant of the medium group are higher than that in the plasma (p<0.001). The content of IL-6 in the supernatant from PBMC stimulated by CpG-302 are much higher than that in the plasma (p<0.001). The stimulatory effects of CpG-302, CpG-2216, CpG-2006 and CpG-C274 are similar (p>0.05). The statistical analysis show: The stimulation of CpG-302 to the cytokine secretion by PBMC does not have remarkable differences between people with and without HBV infection (p>0.05). Patients with different clinical manifestations, different virus markers tested by immunological method, different serum ALT level, different HBV DNA quantities, different age and gender have similar response to CpG-302. The content of IFN-γin the supernatant from PBMC of female patients are much higher than that of the male cases (p<0.05). The content of IFN-γin the supernatant from PBMC of patients with lower HBV DNA quntity are much higher than that of the cases with higher one (p<0.01). The content of IL-12 in the supernatant from PBMC of asymptom HBV carriers are much higher than that of chronic HBV patients (p<0.05). The content of IL-12 in the supernatant from PBMC of patients with positive HBsAb, HBeAb and HBcAb are much higher than that in the patients with positive HBsAg, HBeAg and HBcAb (p<0.05). 6. Analyze the relativity between the antiviral activity and the other effects of CpG-302 The statistical analysis show: There are no obvious relativity between the antiviral activity of CpG-302 and the other effects: stimulation PBMC from people infected with HBV to proliferate and to secret IFN-α, IFN-γ, IL-12 and IL-6 (p>0.05). Taken together, we studied the immunostimulatory activities of CpG-302 using PBMC from people infected with HBV and found the following: 1. CpG-302 can stimulate the proliferation of PBMC from people infected with HBV. The effect of CpG-302 is similar to that of CpG-684, CpG-2006 and CpG-C274. The effect of CpG-302 is obviously strong than that of CpG-2216. The effect of CpG-302 on patients with positive HBsAg, HBeAg and HBcAb is much stronger than that on the patients with positive HBsAb, HBeAb and HBcAb (p<0.05).