Bcg-cpg-dna And Vaccae Adjuvant Hepatitis B Vaccine Immune Response And Its Mechanism

Adjuvant assumes an important role in vaccine development. The immune effects of some traditional vaccines would be weak without adjuvant. Effective adjuvant is an important breakthrough especially for some new or improved vaccines. To date, a wide range of adjuvants are studied by human, but only aluminum hydroxide adjuvant is approved and mainly used in human. MF59 used as adjuvant for subunit influenza vaccine by Novartis Company was approved to enter into Europe market and was authorized to import to China by China FDA in 2007; however, it has not been approved by FDA yet. Cervarix HPV vaccine using AS04 as human adjuvant by GlaxoSmithKline has obtained the patent in UN already, but unauthorized in China.At present, there are lots of studies on adjuvants demonstrating their stronger activity for immunostimulatory activity, but side effects limit their clinical practice In this study, we focused on two adjuvants: BCG-CpG-DNA (for short is CpG) and Vaccae (Mycobacterium Vaccae, for short is Va). The CpG is a kind of natural DNA extracted from BCG and has good immunostimulatory activity as well as safety. As we known, safety is the major bottleneck of adjuvants in the progress to get into clinical trail. Vaccae has a good effect on immunostimulation with respect to the two-way immunoregulation; therefore, there are lots of studies about the treatment of chronic Hepatitis B taking into account the combination of Vaccae and HBV vaccine. Experiments in vitro and in vivo in mice were conducted in this study to discovery the immunostimulatory activity of the two adjuvants initially. Then we mixed the CpG / Va with recombination HBV vaccine (with or without aluminium hydroxide) to find out and compare the immune effects with different adjuvants (mono- vs. combination of adjuvants), and analyze their mechanism of action initially.To discovery the immunostimulatory activity of the two adjuvants initially, we conducted several studies in mice in vitro and in vivo as follow: 1) Used carbon particle clearance assay to object whether the adjuvants affect the function of phagocytic cell. 2) The spleen cells of BALB/c mice were stimulated with two adjuvants in collaboration with ConA in vitro, and the secretion levels of IFN-γand IL-10 in the supernatant of cell culture were determined by ELISA to evaluate whether the immuno-activity could be stimulated by the two adjuvants. 3) We immunized C57BL/6 mice with totally 3 doses of CpG or Va by every 7 days per dose, then we detected the secretion level of IFN-γand IL-10 of spleen lymphocytes by ELISPOT. The proliferation of lymphocyte of spleen was measured by MTT assay after 68 hours in spleen lymphocytes cultured in vitro in order to assess the effects on the immuno activity by CpG or Va.Subsequently, recombinant hepatitis B vaccines (with or without aluminum hydrogen) were administered mice either alone or in combination with CpG or Va, which were classified as N.S. Ag, Va+Ag, CpG+Ag, Al+Ag, Va+Al+Ag, and CpG+Al+Ag. The main steps of experiments were: 1) At different time points of administration, the muscles of injection site, the spleens and the lymphoid nodes were collected to make sections of tissues stained by HE method. Effects of adjuvants on pathological change were evaluated. 2) Analyzing the trends of Time-Content of HBsAg in tissues homogenate and serum, and comparing the different between the groups with or without adjuvant to conclude whether the adjuvant have the function for antigen delivery or slow-release. 3) Change on cellular immune of Th1 lymphocyte in mice immunized with different vaccines was determined by ELISPOT. 4) The antigen-specific antibodies in serum were determined by ELISA and were used to assess the speed of the outcome of humoral immune response, as well as the antibody time-titer trends.The results of immunostimulatory activity of adjuvants showed: 1) There were no statistical significant for the impacts of phagocytosis function with or without BCG-CpG-DNA and Vaccae in mice; 2) BCG-CpG-DNA could induce the secretion of IFN-γand enhanced ConA to stimulate secretion of IFN-γby lymphocyte both in vivo and in vitro. In contrast, Vaccae induced an insignificant level of IFN-γin vivo and in vitro, and could lead to higher level of IL-10 both in vivo and in vitro and suppress ConA to stimulate secretion of IL-10 by lymphocyte, although similar effect to ConA for stimulating secretion of IFN-γ. 3) Tests showed that both adjuvants have effects on cellular immunostimulatory. On basis of above, we could draw a preliminary conclusion that CpG could induce the immunostimulatory activity of Th1 lymphocyte, while Va could incur immuno response of Th1 and Th2 lymphocyte.Moreover, the findings from BCG-CpG-ODN (referred to as CpG) or Vaccae (referred to as Va) used as an adjuvant combining with hepatitis B indicated as follow: 1) CpG and Va could enhance the possibility of inflammatory infiltration of muscle cells in injection site. 2) Although lower effect of antigen release in comparison with AL(OH)3, the two groups of the combination of CpG or Va with HBV vaccine had 2 to 4 times of quantity of antigens in the muscle of injection site than that in the control groups (HBV vaccine alone) after 1 day of vaccination. 3)In addition, both of them had synergism with Al(OH)3: CpG or Va along with Al(OH)3 were more beneficial to the deliver of HBsAg to enter into spleen of mice 4) Hepatitis B vaccine with CpG could induce spleen lymphocyte to secret of IFN-γand IL-2 which induced by Th1 cellular immune responses after 7 days of vaccination; hepatitis B vaccine with Va, on the other hand, could induce a smaller amount of IFN-γand IL-2; Va along with AL could increase the secretion of IFN-γand IL-2 in contrast with either of them; the proportions of positive were promoted after boost 1 dose and were associated with the dose of HBsAg: the group with 3μg had higher positive proportion than that in the group with 1μg after 7 days of administration.5) Each of the combination groups of CpG or Va along with HBsAg could enhance serum antibody titer after administration; Combined with Al(OH)3, CpG or Va was capable of increasing positive serum antibodies, and significantly improving serum antibody titer in 2 to 4 weeks after vaccination.In summary, CpG and Va had the ability of stimulating cellular immune, and played a certain role in increasing the ability of antigen delivery and slow-release after recombinant hepatitis B vaccined. Also, CpG and Va were able to improve cellular immune and increase humoral immune response in mice. Therefore, using CpG and Va as adjuvants in vaccine will be promising.