| Hepatitis B(HB)is one of the sevious diseases harmful to human health.Up to now, no effective drug has been developed yet for chronic HB therapy.Vaccination of hepatitis B vaccine is the only effective measure to control hepatitis B prevalence.The immunogenicity of genetically engineered hepatitis B surface antigen(HBsAg)is low,so adjuvant is needed to induce an effective immune response.Aluminum is the current hepatitis B vaccine adjuvant which has been widely used for many years.However, aluminum adjuvant hepatitis B vaccine is obviously inadequate to prevent or treat hepatitis B.In terms of prevention,aluminum adjuvant hepatitis B vaccine exists about 10 percent of low or no response and in treatment,the efficacy of aluminum adjuvant hepatitis B vaccine is not very clear.Oligonucleotide with BW006 motifs is a new type of immune adjuvant which can stimulate cell proliferation and activation,induce natural and acquired immune response. In present study,technologies of flow cytometry,enzyme-linked immunospot assay, enzyme-linked-immunosorbent serologic assay and branch DNA were used to anlyize the cellular immune activity of BW006.Then we conducte a primary study on the adjuvanticity of BW006 in enhancing the cellular and humoral immune response induced by HBsAg Our study provides an experimental support for the development of new BW006 adjuvant vaccine with independent intellectual property rights for the prevention and treatment of hepatitis B.In the first part,mouse splenocytes are stimulated with BW006 in vitro and in vivo.The results show that:(1)BW006 can effectively increase the expression of surface molecules CD80,CD86 on DC and B-cell in a dose-effect manner.There was a dose-effect relationship;(2)BW006 can activate NK-cell and increase the surface molecules CD69 expression on NK-cell;(3)BW006 can inhibite B-cell apopotosis and promote B-cell proliferation;(4)After immunization with BW006,the best time to detect the activation of splenocytes:DC for 12 hours,B,NK and T cells for 24 hours;(5)The order of B-cell's activation induced by different immune channels of BW006 is muscle immune＞abdominal immune＞subcutaneous immunization;(6)Chloroquine phosphahate can inhibit the role of BW006 activates DC,B and NK cells.Branch DNA technology was used to analysis of the cytokine mRNA level expressed by mouse spleen mononuclear cells(MNC)and DC that stimulated by BW006 in different time.The result showed that the level of IL-12b mRNA expressed by splenocytes is obviousely higher than other cytokine mRNA.The peak of IL-12b mRNA expression is between 3 and 6 hours after stimulating by BW006.Compared with control, IL-12b mRNA expression level increased by 26 times and IL-10 mRNA by 11.9 times after 6 hours stimulating by BW006.There was a difference between MNCs and DC in IP-10 mRNA expression.Three hours late stimulating by BW006,mouse spleen MNCs and DC IP-10 mRNA expression levels were increased by 2.8 times and 7.26 times respectively.Luminex technology was used to detect the dynamic process of mouse spleen MNCs IL-12 and IL-10.The result showed that BW006 in vitro stimulation can increase mouse spleen MNCs IL-12,IL-10 expression,and showed the time-effect relationship. With the BW006 stimulating time extension,IL-12 expression level increased gradually. The peak time of IL-12 is 48 hours,and 72 hours to maintain the same level.Expression trend of IL-10 is similar to IL-12.With the BW006 stimulating time extension,IL-10 expression level also increased gradually and the peak time is 48 hours.But after 48 hours,IL-10 expression levels decreased gradually.In addition,3 hours after immunization with 200μg BW006 through subcutaneous,we use the ELISA method to detect mouse peripheral IP-10 level.The result showed that peripheral IP-10 expression level significantly increased.Compared with the control group,there was an increase of 46.55 times(p＜0.01).In the second part BW006 was investigated for the feasibility of enhancing HBsAg cellular and humoral immune response.Firstly,we explored the best time and dose of DC activation induced by HBsAg in vitro.The best time is 24 hours and the best dose is 40μg. Stimulating mouse splenocytes with different doses of BW006 joint 40μg HBsAg in vitro, DC expression of CD80,CD86 levels increased significantly.There were dose-effect relationship.In vivo results also showed that BW006 and HBsAg can collaboratively induce B and T cells activation.Secondly,we immunized BALB/c mouse with different dose of BW006 joint 2μg HBsAg or 2μg hepatitis B vaccine.Seven days after immunization,enzyme-linked immunospot assay(ELISPOT)was conducted to analysisi of mouse spleen cells secreting IFN-γand IL-2 level.The result suggested that:(1)BW006 can significantly improve HBsAg or aluminum adjuvant hepatitis B vaccine to induce immune cells secreting IFN-γand IL-2;(2)The expression levels of IFN-γand IL-2 induced by BW006 joint hepatitis B vaccine were significantly lower than BW006 joint HBsAg;(3) The best dose of IFN-γexpression induced by BW006 joint HbsAg or hepatitis B vaccine is 1.25μg.However,the best dose of IL-2 expression induced by BW006 joint HbsAg or hepatitis B vaccine is 5μg.Finally,we compared the humoral immune response levels induced by BW006 joint HBsAg and BW006 joint hepatitis B vaccine.Balb/c mice were immunized with different dose of BW006 joint 2μg HBsAg or 2μg hepatitis B vaccine.Antibodies were detected 1,2,3 and 4 weeks.The results showed that:(1)BW006 joint HBsAg or hepatitis B vaccine in m ice c an increase a nti-HBs response l evels;(2)Since the third week after immunization,the antibody response levels induced by BW006 joint hepatitis B vaccine were significantly higher than BW006 joint HBsAg.(3)The best dose of BW006 joint HBsAg inducing humoral immune response is 5μg.However,the best dose of BW006 joint hepatitis B vaccine inducing humoral immune response is 20μg.In short,we analyzed the cellular immune activity of BW006 in vitro and in vivo. The feasibility of BW006 as HBsAg adjuvant was explored from natural immunity, specific humoral immunity and specific cellular immunity.We found that BW006 can effectively activate immune cells and coordinate HBsAg or hepatitis B vaccine inducing stronger humoral and cellular immune response.Our study can provide a new way of thinking for research and development of new hepatitis B vaccine. |
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