Effect Of Synthetized Glucohexaose On The Immune Response Induced By Hepatitis B Virus Antigen

Effect of synthetized glucohexaose on the immune response induced by hepatitis B virus antigenHepatitis B virus (HBV) is a major pathogen of acute and chronic hepatitis. HBV infection may result in acute or chronic hepatitis, and may eventually develop to cirrhosis and hepatocelluar carcinomar. HBV belongs to hepadnaviruses family and it is primarily hepatotroPic virus and only infects human and chimpanzee, but not non-primate laboratory animals. Despite the development and extensive use of vaccines, hepatitis B virus infection remains an important problem to human health worldwide. In fact, more than 5 million people world-wide become infected by hepatitis B virus each year, and the WHO has reported over 350 million carriers of the virus. Vaccination with hepatitis B surface (HBsAg) protein has been used for many years to induce protection against HBV infection, which correlated with the induction of neutralizing antibodies (anti-HBs). Though recognized as an effective vaccine, nearly 10% of vaccinees with HBsAg fail to develop protective levels of anti-HBsAg antibodies. DNA vaccine could induce antigen-specific cytotoxic T lymphocytes, but it has relatively low efficacy in large animal species and humans. Although it induces low immune response in clinic trial, DNA vaccine has a great future in the protection and therapy of some diseases. The main point of the failure in HBV vaccine is to induce low immune response. Thus, approaches to improve vaccination efficacy are actively sought.Ganoderma lucidum and other edible Mushroom have been used for a long time in China to prevent and treat various diseases. Polysaccharides of fungal origin have important immunostimulatory effects, acting mainly in the activation of macrophages and dendritic cells through selective innate recetors (such as dectin-1). Recent pharmacodynamic characterisation of the active principles in such extracts has revealed that many polysaccharides and oligosaccharides are among the bioactive molecules. Among the best characterized bioactive polysaccharides has been shown to have potent anti-tumour activity due to its powerful capacity to stimulate cell mediated immune responses and to activate innate immune effector cells such as monocytes/macrophages and NK cells and DC. It also shows that polysaccharides have anti-inflammatory activity by promote the production of anti-inflammatory factor and inhibit the production of inflammatory factor. Besides for the anti-tumour, immunomodulator and anti-inflammatory capacity of polysaccharides, it shows protective effect on the autoimmune disease. Possibly because of its ability to stimulate immune response, polysaccharides, as an adjuvant, were found able to reduce tuberculosis infection and to enhance the effect of BCG vaccination and to enhanced the immune response induced by HBV DNA vaccine. In addition, they can induce T cells immune response by T cell independent and T cell dependent way according to their structure. Indeed, the use of oligosaccharidic adjuvants appears very promising for targeted stimulation of innate immunity and could help to biasing the immune response towards more effective protection.β-(1→6)-branchedβ-(1→3) gluco-oligosaccharides are the common structural moiety of many bioactive polysaccharides from plants and fungi. The anti-tumour activity of lentinan, theβ-(1→3)-D-glycan with 2β-(1→6)-glucosyl branches every 5 residues, appears to be related to its structure and molecular weight. The triple-helix conformation of the backbone chain in lentinan is important for the biological effect, and the basic unitβ-(1→6)-branchedβ-(1→3) glucohexaose is believed to play a major role in anti-tumour activity. Indeed, theβ-(1→6)-branchedβ-(1→3)-glucoexahose and its analogues withα-(1→3)-linked bond show similar effects as lentinan in anti-tumour activity in the experimental S 18 tumour model and in amplification of spleen cell proliferation and induction of TNF-αproduction. On the other hand, theβ-glucan mainly containing 1, 6 linkages are significantly less active. Thus, it appears that the basic unit ofβ-glucan is endowed with the immunostimulatory effects of the whole polysaccharide. With the similar effecter with polysaccharides and the simple structure, high purity and high yield of synthetized glucohexaose, it attracts more attention on the synthesis and bioactivity of glucohexaose. Based on the structure unit of lentinan, theβ-(1→6) branchedβ-(1→3) glucohexaose analogue which containing anα-(1→3)-linked bond (named asβ-glu6 in this paper) was sythetized by professor Gu group. The immunomodulatory activity and adjuvant capacity ofβ-glu6 was examined in vivo and vitro in this thesis.Part I Effect ofβ-glu6 on the activation of marrow dendritic cellsIn order to test whether it contains contaminants and the purity of theβ-glu6, theβ-glu6 was tested by HPLC and LPS detection and it showed that the purity was more than 98% and without LPS contaminate.β-glu6 showed no cytotoxicity in this study and it promoted the proliferation of cells in some extent. According to the reports, marrow dendritci cells (MDC) were differentiated from bone marrow cell with stimulation of IL-4 and GM-CSF and treated with different concentration ofβ-glu6 in vitro. The expression of co-stimulate molecular were detected by flow cytometry and results showed thatβ-glu6 enhanced the expression of CD40, CD80 and CD86 (50.85%, 46.19% and 39.87%, respectively; vs 42.85%, 39.78% and 27.81%, mock, respectively ). It also promoted the maturation of MDC by 10%-16% examined by flow cytometry for the percentage of CD11 c~+ cells co-expressing CD40, CD86, CD80 or MHC classⅡ. In the meantime, the maturation of MDC induced by HBsAg was enhanced by 12%-26% withβ-glu6 treatment. However, there are no effects on the phagocytosis of MDC. The migration of mature MDC was detected and it showed thatβ-glu6 promoted the migration of mature MDC to local lymph node. The percentage of migrated MDC in right inguinal lymph nodes was 2.07%, which was about 20-fold increasing compared with untreated cells, but there are no effects on the expression of IL-10. Been migration to local lymph node and to interaction with T cells to induce T cells immune response of maturation of MDC, mixed lymphocyte reaction of MDC was detected and the result showed that the proliferation of T cells was increased induced by MDC with the treatmentβ-glu6 and HBsAg. In this part, it showed thatβ-glu6 enhanced the function of MDC.PartⅡEffect ofβ-glu6 on the activation of RAW264.7 cellsMacrophages can be stimulated by various agents such as LPS to produce TNF-a, IL-1, and IL-6. These cytokines play an important role in various immune responses and in inflammatory reactions. Apart from LPS, a wide variety of polysaccharides of microbial and plant origins are also known to stimulate immune system including activation of macrophage functions. In particular,β-D-glucans from fungi have been reported to induce secretion of inflammatory cytokines from macrophages, and some of these polysaccharides are known as biological response modifiers with apparent anti-tumor activity. We first examined the proliferation activity RAW 264.7 cells with treatment ofβ-glu6 by MTT assay. The results showed thatβ-glu6 promoted the proliferation of RAW 264.7 cells with 100μtg/ml. Phenotypes of macrophage were analyzed using flow cytometry. In comparison with untreated macrophages, the expression of surface molecules was detected, including CD40, CD80, CD86, MHCⅠand MHCⅡ, only CD40 was increased onβ-glu6 stimulated macrophages, and there are no effect on CD80 and MHCⅡ, but decreased the expression of CD86 and MHCⅠmolecular. Endocytosis of macrophage was analyzed by the uptake of HRP and neutral red (NR). In RAW 264.7 cells treated withβ-glu6, the endocytosis ratio of HRP and NR were 637% and 40.2, which about 1.5-2 fold increased compared with untreated cells. Then we investigate whether the TNF-αproduction was increased in macrophage stimulated withβ-glu6. In control RAW264.7 cells in the absence of oligosaccharides, only small amounts of TNF-αwas secreted into the medium (260pg/ml). However, in the presence ofβ-glu6, the secreted levels of TNF-αincreased to 336pg/ml. To examine the possible involvement of TLR2 and TLR4 inβ-glu6 induced TNF-αsecretion from RAW264.7 cells, the specific blocking antibodies were used. As results shown, both antibodies to TLR2 and TLR4 inhibited the activities ofβ-glu6 to induce TNF-αsecretion. In this part experiment, the results showed that there are no effects ofβ-glu6 on the production of IL-10, a down-regulator of macrophage and the member of Th2 cytokines. In this part, it showed thatβ-glu6 enhanced the function of macrophage.PartⅢEffect ofβ-glu6 on the production of IL-18 and relative eytokines in human monoeyte and HT29 cellsInterleukin-18 (IL-18), a recently described member of the IL-1 cytokine superfamily, is now recognized as an important regulator of innate and acquired immune responses. IL-18 is expressed at sites of chronic inflammation, in autoimmune diseases, in a variety of cancers, and in the context of numerous infectious diseases. To investigate whether the IL-18 production was affected in human monocyte and HT29 cells stimulated withβ-glu6, these cells were treated withβ-glu6 in vitro and the expression of IL-18 was measured by Real-time PCR. The results showed that IL-18 expression was 0.5 fold decreased with the treatment ofβ-glu6 compared with untreated cells. There is no true receptor antagonist to regulatory activity similar to IL-1Ra. Conversely, IL-18 regulation is apparently carried out by a soluble IL-18-bingding protein (IL-18BP), which binds IL-18 preventing its interaction with the membrane IL-18R. Therefore, we investigate whether the effect ofβ-glu6 on the production of IL- 18 was increased the production of IL-18BP. As results shown, the IL-18BP expression was 1.32±0.15 in human monocyte and 2.52±0.54 in HT29 cells, which have 1.3-2.5 fold increasing compare with untreated cells. The ratio between IL-18BP and IL-18 were increased with the treatment ofβ-glu6. It showed that the ratio in human monocyte was 2.98 and 4.49 in HT29 cells, with about 3-4.5 fold increasing in contrast with untreated cells. TIR8 is a unique receptor of the IL-1R/IL-18R superfamily and apparently plays a central role in the down-regulation of inflammation mediated by TIR receptors. In our experiment, we found thatβ-glu6 enhanced the expression of TIR8 from 1.01±0.13 (untreated cells) to 1.74±0.16 (treated cells) in HT29 cells. On the contrary,β-glu6 decreased the expression of TIR8 from 1.04±0.1 (untreated cells) to 0.63±0.14 (treated cells) in human monocyte. Interestingly,β-glu6 decreased the IL-18BP and TIR8 expression induced by IL-1β. To investigate whether the inhibit effect ofβ-glu6 on IL-1βwas due to the increasing of IL-1Ra, IL-1Ra was measured with Real-time PCR. The data showed that,β-glu6 and IL- 1βenhanced the expression of IL-1Ra, on the contrary, it was decreased co-treated withβ-glu6 and IL-1β. Then we detected the expression of IL-18R, the results showed that there are on effects ofβ-glu6 on the expression of IL-18Rβ, but it enhanced the expression of IL-18Rα.PartⅣEffect ofβ-glu6 on the immune response induced by hepatitis B virus surface antigenThe syntheticβ-(1→6)-branchedβ-(1→3) glucohexaose and its analogues, the basic unit of lentinan and otherβ-glucans, has been reported to have immunostimulatory effects comparable to those of natural lentinan. In this part, we investigate the adjuvant effect ofβ-glu6 on the immune response induced by HBsAg. BALB/c mice were immunized with HBsAg andβ-glu6. Compared to untreated or PBS-treated control mice, immunization with HBsAg induced a significant influx of F4/80~+ cells, already evident 3 days after antigen administration (5.1-fold increase) and further increasing after the second antigen inoculum (8.7-fold increase at day 35), The use ofβ-glu6 as adjuvant in the immunisation with HBsAg rapidly and dramatically increases the influx of macrophages in the spleen, with over 10-fold increase at day 3 after the first inoculum, and over 14-fold increase after the second inoculum. co-administration ofβ-glu6 together with the antigen greatly induces migration of DC to the spleen in response to HBsAg (13.5×10~6 CD11c~+ cells/spleen; 5.6-fold increase vs. untreated, over 2-fold vs.β-glu6 alone, and 1.5-fold increase vs. HBsAg alone). Maturation of recruited DC was more pronounced in spleens of mice immunized with HBsAg together withβ-glu6, as compared to cells from animals receiving antigen alone or antigen with the Freund's adjuvant. Administration ofβ-glu6 alone had little but significant effect, in particular in increasing the number of CD40~+ DC. 35 days after primary immunization,β-glu6 could significantly amplify the HBsAg-induced recruitment/proliferation of CD4~+ T cells in the spleen, while the sugar or the antigen alone had lower effect. Similar results were obtained in the liver, where influx of CD4~+ T cells was significantly higher in mice treated withβ-glu6 together with HBsAg as compared to antigen or adjuvant alone. In the spleen, the number of activated CD4~+ T cells was significantly increased in mice immunized with HBsAg together with the sugar (11.8×106 CD4~+CD69~+ cells/spleen). Six weeks after primary immunisation (i.e., two weeks after boost), spleen cells were collected, stimulated with HBsAg in culture, and analysed with ELISPOT for the specific antigen-driven production of IFN-γand IL-4. Results showed that in spleen cultures from mice immunized with HBsAg alone the in vitro stimulation with the specific antigen could induce a significant number of cytokine-producing cells (2,762 IFN-γ~+ cells/spleen vs. 148 in untreated spleens; 8,437 IL-4-producing cells vs. 404 in control spleens). However, in mice immunized with HBsAg together withβ-glu6, the number of HBsAg-specific IL-4-producing cells was dramatically increased by antigen stimulation (30,058 IL-4~+ cells from mice immunized with HBsAg plusβ-glu6 vs. 8,437 with HBsAg alone). This increase was selective for IL-4-producing cells, as the number of IFN-7-producing cells was not significantly affected. Five weeks after primary immunization, the number of activated B cells is significantly increased in the spleen of mice immunised with the HBsAg alone and further enhanced when the antigen is administered withβ-glu6 as adjuvant. To evaluate the effect ofβ-glu6 in enhancing the HBsAg-specific antibody response, serum antibody titers were measured at different times after primary immunization, from 4 weeks up to 12 weeks. Mice immunized with HBsAg without adjuvant developed significant antibody response only after the second antigen inoculum (i.e., from 6 weeks onwards), which however remained at low levels (titers between 1/800 and 1/3600). Conversely, mice receiving the antigen together withβ-glu6 adjuvant showed low but significant antibody levels already 4 weeks after primary immunization (1/300), which were highly increased by the second challenge (titers＞1/25,000). After 12 weeks, anti-HBsAg titers still persisted at high levels (1/7000). The IgG1 subclasses and the ratio between IgG1 and IgG2a induced by HBsAg were increased byβ-glu6, but there are no effects on the IgG2a subclasses. In this part, it showed thatβ-glu6 enhanced the Th2 immuneresponse induced by HBsAg. PartⅤEffect ofβ-glu6 on the immune response induced by DNA vaccine encoding hepatitis B virus core antigenBeing inducing antigen-specific cytotoxic T lymphocytes, DNA vaccine had been investigate extensively in protection and therapy of disease. In this part, we observed the effect ofβ-glu6 on the immune response induced by DNA vaccine encoding HBV core antigen (pB144). Mice were immunized withβ-glu6 and pB144 and the immune response was measured in different time point. Maturation of recruited DC was more pronounced in spleens of mice immunized with pB144 together withβ-glu6, as compared to cells from animals receiving pB144 alone. 5 and 35 days after primary immunization,β-glu6 could significantly amplify the HBsAg-induced recruitment or proliferation of CD8~+ T cells in the spleen, liver and PBMC. Interestingly, we also found thatβ-glu6 enhanced the recruitment of CD4~+T cells in spleen, liver and PBMC. In the spleen, 5 days and 35 days after primer immunization, the number of activated CD8~+ T cells was significantly increased in mice immunized with pB144 together with the sugar (5.92×10~6 and 8.31×10~6 CD8~+CD69~+ cells/spleen). The increasing of CD4 activation also observed at day 5 and 35. In the spleen, the number of activated CD4~+ T cells was significantly increased in mice immunized with pB144 together with the sugar. In pB144 immunized mice, the CD4~+CD69~+ cells were 3.19×10~6 cells/spleen. However, it showed 1.5 fold increasing with the treatment of pB144 and sugar. At day 5, 14 and 40, the antigen specific CD8~+T cells immune responses were measured with intracellular cytokine staining. At day 5 and 14, there are no effect ofβ-glu6 on the antigen specific CD8~+T cells. After booster immunization, the antigen specific CD8~+T cells were significant increased. At day 40, the antigen specific CD8~+T cells in mice treated with pB144 were 0.8×10~6 cells/spleen. However, the number of antigen specific CD8+T cells was 1.5×10~6 cells/spleen in pB 144 andβ-glu6 co-immunization mice, with about 2 fold increasing compare with pB144 immunization mice. It showed the similar effect ofβ-glu6 in PBMC and liver. At day 40, the percentage of antigen specific CD8~+T cells in PBMC and liver of pB144 treated mice were0.38%±0.12% and 0.22%±0.04%, but it was 2 fold increasing co-immunized withβ-glu6 (0.74%±0.17% in PBMC and 0.54%±0.08% in liver). To evaluate the effect ofβ-glu6 in enhancing the HBsAg-specific antibody response, serum antibody titers were measured at different times after primary immunization, at 4 week and 6 week. Mice immunized with pB144 without adjuvant developed low antibody at 4 week (1/900), which however increased at 6 week (1/8100). Conversely, mice receiving the antigen together withβ-glu6 adjuvant showed low but significant antibody levels at 4 weeks after primary immunization (1/2700), which were highly increased at 6 week (titers＞1/24,000). Interestingly, the IgG1 subclasses in every group were undetectable, but the IgG2a subclasses and the ratio of IgG2a/IgG1 were increased with the co-immunization of pB144 andβ-glu6. All of these data mean thatβ-glu6 enhanced the immune response induced by pB144.