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Effect and Its Mechanisms of Hepatitis B Virus Antigens Conditioned with a TLR7/8Agonist for Chronic Hepatitis B Infection

Author: WangYing
Tutor: QuChunFeng
School: Beijing Union Medical College
Course: Immunology
Keywords: TLR7/8agonists Monocyte-derived DCs follicular helper T cells therapeutic vaccine HBV
CLC: R512.62
Type: PhD thesis
Year: 2013
Downloads: 1
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Abstract


Chronic persistent hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma in China. Clearance of HBV infected liver cells may block the progression of hepatocellular carcinoma. Currently, antiviral therapies have been proved to be efficient for most of the chronic HBV infection. However, they fail to eradicate HBV infected liver cells due to the persistence of HBV covalently closed circular DNA(cccDNA) in hepatocytes and the emergence of resistant viruses. Moreover, long-term treatment is expensive and may result in problems of toxicity and intolerance. These limitations of antiviral therapies of HBV underline the need for alternative therapeutic approaches. HBV therapeutic vaccine is a promising new strategy for controlling chronic infection due to its effective activation of antigen-presenting cells to uptake and present vaccine antigens, and then induce and maintain a potent humoral and cellular immune response.Activation of Toll-like receptors (TLRs) on immune cells shapes the immune responses to different types of antigens. In clinical vaccine research some TLR ligands have been used as adjuvants. Previously, we reported monocytes-derived dendritic cells (Mo-DCs) played an important role in the induction of antigen-specific immune responses to particulate antigens, and TLR8agonists stimulated newly recruited monocyte-derived cells into potent APCs that enhanced HBsAg immunogenicity. Comparing with the vaccine with aluminum adjuvant alone, TLR7/8conditioned vaccine induced a stronger anti-HBsAg response in mice. Hepatitis B core antigen (HBcAg) is the HBV structural antigens. Specific cellular immunity to HBcAg were reported to be involved in stopping HBV infection in patients. Some studies have reported that physical association of TLR agonists to targeted antigen is required in order to induce efficient antigen presentation, thus stimulating the production of strong Th1and CD8+T cells. In this study, we prepared an HBV vaccine containing5μg HBsAg,5μg HBcAg and5μg CL097(TLR7/8agonist) conjugated with alum hydroxide. By using UPLC-Q-TOF-MS we confirmed that CL097was absorbed to aluminum with HBV antigens to form particle antigen. HBV-Ag and TLR7/8agonist were physically associated with alum hydroxide gel through surface charge. In order to trace the cells that could take this kind of vaccine antigens HBsAg were then labeled with FITC adsorbed to alum with or without CL097added in the vaccine formula and administered into the mouse skin via intradermal injection. Forty-eight hours later, draining lymph nodes were collected and analyzed by FACS. In the presence of CL097, more FITC-positive DCs were found in the lymph nodes than that with alum alone. Collecting the cells in the injection sites18hours later, it was found that antigens were mainly taken up by CD1lb+(≥90%)cells but not the CDllc+(<10%) cells. Cell populations that took up the antigens showed no significant difference with or without CL097included in the formula. Among the CD11b+cells, around60%of the cells showed Ly6c high expression and40%of them showed low or no Ly6c expression. We then sorted the antigen positive and CD11b+Ly6c" cells and CD11b intLy6c+cells injected back into the mouse skin. The two population cells could differentiate into CD11c+cells and carried the antigen into draining lymph nodes. We then isolated the Ly6c+from mouse splenocytes and stimulated with HBV-Ag vaccine with or without CL097conditioned. Three days later, compared with the antigen adsorbed to alum alone more IL-6and TNF-a were found in the supernatant, more monocytes differentiated into CD11c+DC. All the cells expressed higher levels of CCR7and CD86. The stimulated monocytes were futher co-cultured with naive T cells for7days. T cells expressed higher level of BCL-6and T-beta showing a characteristics of the follicular helper T cell with higher CXCR5expression and IL-21, TNF-a and IFN-y secretion.We immunized two different strain HBV transgenic mice, mimicking human chronic HBV infection, with the CL097conditioned HBV-Ag vaccine every two weeks to determine its therapeutic effect. Two weeks after the fourth immunization, anti-HBs was induced in54.5%(6/11) of the mice, with the concentration of85.88mIU/ml. The response to the vaccinated protein lasted for more than20weeks once the immune responses generated. HBsAg-specific B cells, IFN-y-producing CD4+/CD8+T cells and IL-21+T cell could be found in the splenocytes of mice immunized with CL097conditioned HBV-Ag vaccine. The above results suggest that this formula vaccine induced HBsAg-specific humoral and cellular immune responses in HBV transgenic mice.Then we sorted the CD4+CD25+Treg cells and Treg-depleted cells from the mice immunized with CL097conditioned HBV-Ag vaccine and transferred into un-immunized normal C57BL/6J mice. Anti-HBs antibodies could be detected3days after immunization with5μg HBsAg. After adoptive transfer of CD4+CD25+cells Treg, specific antibody could also be detected12days after the immunization. In contrast, after transfer of these two populations from non-immunized HBV-Tg mice into unimmunized normal C57BL/6J mice, no specific antibodies were detected for more than12days. CL097conditioned HBV-Ag vaccine broke the specific immune tolerance and induced the antigen-specific immune responses in vivo. Combination of IFN-a-2b with this kind of vaccines was also observed in the HBV-trangenic mice. Injection of IFN-a-2b for3times before the vaccination could accelerate the generation of anti-HBs.We also established a syngeneic mouse model of liver tumor stably expressing HBV antigens. B16/HBV cells which persistently secreted HBV antigens were injected into the liver of C57BL/6J mice beneath the capsular after immunized with CL097conditioned HBV-Ag vaccine for3times. Compared with the mice that immunized with commercialized prophylactic vaccines, the mice immunized with CL097condition HBV-Ag vaccine extended5more days of survival. One of the7treated mice survived up to70days. Antigen-specific IFN-y secreting T cells were detectable in the splenocytes.Summary:1. TLR7/8agonist CL097and HBV-Ag were physically associated with alum hydroxide gel through surface charge to form particulate antigens. This kind of vaccine antigens were mainly phagocytosed by CD11b+monocytes after intradermal injection. CL097promoted their maturation and differentiation. In the presence of CL097more antigen carrying cells homed to draining lymph nodes;2. The monocyte-derived DC stimulated by TLR7/8agonist conditioned HBV-Ag vaccine could promote naive T cell differentiation into CXCR5-positive follicular helper T cells, producing IL-21.3. In the HBV transgenic mice which mimic chronic HBV infection status, immunization with TLR7/8agonist conditioned HBV-Ag vaccine broke the HBV-specific immune tolerance and induced anti-HBV immune reposes. Combination of IFN-I with this kind of vaccines accelerate the generation of anti-HBs. Therefore, immunization with TLR7/8agonist conditioned HBV-Ag might be a potential approach for treatment of chronic HBV infection.4. Immunization with TLR7/8agonist conditioned HBV-Ag vaccine could induce strong humoral and T cell immune responses in normal C57BL/6J mice. The specific immune responses delayed the tumor cell growth in liver that expressed HBV-antigens.

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CLC: > Medicine, health > Internal Medicine > Infectious disease > Viral infections > Viral Hepatitis > Hepatitis B
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