【作者】 吕树娟；

【导师】 魏海明；

【作者基本信息】 中国科学技术大学 ， 细胞生物学， 2014， 博士

【摘要】 慢性乙型肝炎病毒(HBV)是危害人类健康的难题之一,目前全球至少有3.6亿乙型肝炎病毒感染者,其中超过三分之一的人群在中国,这些人群面临着发展为肝纤维化、肝硬化甚至肝癌的高风险。由于慢性乙型肝炎患者对病毒产生不同程度的免疫耐受,以及HBV共价互补环状DNA(cccDNA)在肝内的持续存在,现有的抗病毒药物如干扰素(IFN)和拉米夫定等核苷类似物,只能一定程度抑制HBV的复制,最终达不到彻底清除HBV的效果,而且尚有一大部分慢性乙肝患者对上述抗病毒治疗无应答,因此发展更为有效的抗病毒药物是十分迫切。通常情况下,病毒感染后会通过一系列机制激活免疫系统从而被清除,特别是病毒感染早期诱导表达Ⅰ型干扰素(IFN-α/IFN-p),I型干扰素不仅能诱导大量的抗病毒蛋白,而且可以激活固有免疫和适应性免疫系统从而发挥抗病毒作用。浆细胞样DC (Plasmacytoid dendritic cells,pDCs)是诱导干扰素-α(IFN-α)表达的主要天然免疫细胞,pDCs通过其胞内的TLR样受体(Toll-like receptors,TLR)包括TLR7和TLR9分别识别病毒的单链RNA和非甲基化的CpG (cytidine phosphate guanosine oligodeoxynucleotides) DNA从而启动下游级联信号,大量分泌Ⅰ型干扰素。而HBV是一种双链DNA病毒,在其基因组上我们发现也存在CpG序列,那么HBV是否可以通过自身CpG触发TLR9信号从而启动下游干扰素活化途径进而清除病毒呢?基于以上问题考虑,本研究首先筛选HBV基因组来源的CpG,即HBV-CpG,探讨其诱导IFN-α表达的能力,在研究过程中还发现HBV基因组中存在富含鸟苷的抑制性寡核苷酸(ODN),即HBV-ODN,这些抑制性HBV-ODN可特异性抑制HBV-CpG诱导的IFN-α表达。进一步研究发现纳米材料PEG-PLA包裹的HBV-CpG,即NP(HBV-CpG),能拮抗HBV-ODN对IFN-a产生的抑制作用,同时探讨了NP(HBV-CpG)在预防和治疗乙型肝炎病毒感染中的作用。在本研究中,我们首先设计了一系列HBV基因组来源的CpG序列即HBV-CpG,利用ELISA和流式细胞术的方法筛选出能诱导健康人PBMCs高表达IFN-α的HBV-CpG;之后,我们设计了HBV基因来源的富含鸟苷的抑制性HBV-ODN, ELISA方法验证其对HBV-CpG诱导IFN-α表达的能力具有特异性抑制作用,免疫荧光技术探讨了抑制性HBV-ODN的抑制机制；双乳化法将纳米材料包裹HBV-CpG形成NP (HBV-CpG), ELISA方法验证NP(HBV-CpG)逆转抑制性HBV-ODN介导的抑制活性,ELISA和流式细胞术进一步检测NP(MBV-CpG)诱导乙肝病人PBMCs表达IFN-a的情况；在小鼠模型中,利用流式细胞术检测野生型小鼠淋巴细胞的活化情况,放射性免疫法及ELISA法检测NP(HBV-CpG)联合乙肝疫苗接种野生型小鼠后的抗体应答；利用高压注射pAAV/HBV1.2质粒构建HBV携带小鼠模型,流式细胞术和ELISA法检测淋巴细胞的活化以及Ⅰ型IFN的表达,放射性免疫法检测NP(HBV-CpG)联合乙肝疫苗治疗后HBsAg和HBsAb的表达,荧光定量PCR技术检测联合治疗后HBVDNA的表达情况,免疫组化法检测联合治疗后肝脏HBcAg表达情况,H＆E染色、转氨酶及胆红素检测观察肝脏损伤情况,流式细胞术内标IFN-γ检测特异性CD8+T细胞的活化。通过上述研究方法,我们获得了以下研究结果：1. HBV-CpG诱导人pDCs细胞高表达IFN-a首先我们设计了一系列HBV基因组来源的CpG序列,将其分别刺激健康人PBMCs, ELISA检测上清IFN-α的表达,由此筛选出高表达IFN-α的HBV-CpG,进一步流式细胞术验证HBV-CpG是通过诱导Lineagel-, CD123+, HLA-DR+的细胞即pDCs特异性表达IFN-α,流式内标发现HBV-CpG刺激PBMCs后pDCs胞内TLR9表达上调,TLR7表达没有显著变化,内体酸化抑制剂氯喹处理后抑制了HBV-CpG诱导的IFN-α的表达。以上结果证明了HBV-CpG诱导人pDCs表达IFN-α,依赖TLR9而非TLR7的途径。2. inhibitory HBV-ODN特异性抑制HBV-CpG诱导的IFN-a的表达在HBV基因组上我们同时发现了一些富集鸟苷的序列,ELISA法证明这些序列具有特异性抑制HBV-CpG诱导IFN-a表达的能力,该抑制性序列命名为HBV-ODN。我们也发现这些抑制性HBV-ODN并不能抑制广谱CpG-2216诱导的IFN-a的表达,为了进一步证明HBV-ODN的抑制机制,我们应用免疫荧光技术发现HBV-ODN抑制了Gen2.2细胞对HBV-CpG的摄取及与TLR9的共定位。因此我们认为抑制性HBV-ODN特异性抑制了HBV-CpG诱导的IFN-a的表达。3. NP(HBV-CpG)逆转HBV-ODN抑制IFN-α的表达能力为了逆转HBV-ODN的抑制效应,我们采用纳米材料PEG-PLA包被HBV-CpG,即形成NP(HBV-CpG),当NP(HBV-CpG)与HBV-ODN以2：1的比例刺激PBMCs时,IFN-α的表达显著提高,而且NP(HBV-CpG)比HBV-CpG具有更强的活化pDCs、NK和T细胞的能力,进一步研究发现NP(HBV-CpG)同样可以诱导乙肝患者来源的PBMCs表达IFN-α。根据以上结果我们证明HBV-CpG在纳米材料包被下可以逆转HBV-ODN抑制IFN-a的能力并且可以诱导乙肝患者PBMCs表达IFN-α。4. NP(HBV-CpG)活化野生型小鼠的淋巴细胞小鼠体内试验中,通过静脉注射NP(HBV-CpG)可以显著提高pDCs和cDCs表面CD40和CD80的表达,以及上调NK和T细胞表面CD69和ICOS的表达。NP(HBV-CpG)体外刺激小鼠脾脏淋巴细胞,同样提高了pDCs和cDCs表面CD40和CD80以及NK和T细胞表面CD69的表达,而且pDCs和cDCs在NP(HBV-CpG)刺激下生存能力增强。以上结果证明NP(HBV-CpG)体内体外均具有活化小鼠淋巴细胞的能力。5. NP(HBV-CpG)协同增强乙肝疫苗的抗体应答NP(HBV-CpG)联合rHBsAg疫苗分别免疫BALB/c和C57BL/6小鼠,同时设空载体联合rHBsAg疫苗组、单独乙肝疫苗组和未处理组做对照,免疫2和4周后放射性免疫法检测发现,NP(HBV-CpG)联合rHBsAg疫苗组抗体应答水平显著提高,ELISA检测发现Thl型抗体亚型IgG2a水平也显著增强。上述结果证明NP(HBV-CpG)增强乙肝疫苗的抗体应答特别是Th1型抗体应答。6. NP(HBV-CpG)促进HBV携带小鼠的免疫应答将pAAV/HBV1.2质粒高压注射C57BL/6小鼠,构建HBV携带鼠模型,NP(HBV-CpG)静脉处理HBV携带鼠显著提高了cDCs表面CD40和CD80, NK细胞表面CD69和ICOS,以及CD4+和CD8+T细胞CD69的表达,并且NP(HBV-CpG)尾静脉注射HBV携带鼠诱导了血清中IFN-α的表达。以上结果证明NP(HBV-CpG)具有活化HBV携带鼠免疫应答的能力。7. NP(HBV-CpG)联合治疗有效清除HBV利用上述高压注射构建的HBV携带鼠模型,探讨NP(HBV-CpG)对于乙肝病毒的清除作用,将NP(HBV-CpG)联合rHBsAg疫苗治疗HBV携带鼠,经过连续三周的治疗,90%的HBV携带鼠血清HBsAg被清除,肝脏HBcAg和血清HBVDNA表达显著下降,保护性乙肝抗体HBsAb开始表达,并且表达IFN-y的CD8+T细胞上调,提示NP(HBV-CpG)促进了CTL功能,同时肝脏H&E染色显示肝脏结构正常,仅有轻微炎性浸润,血清转氨酶和胆红素检测进一步证明HBV携带鼠对这种NP(HBV-CpG)联合治疗是耐受的。根据以上结果我们证明了NP(HBV-CpG)联合治疗HBV携带鼠可以在短期内有效清除HBV,并且促进保护性抗体的产生。更多还原

【Abstract】 Persistent infection with the HBV (Hepatitis B Virus) has become a severe public health problem, and more than360million people worldwide affect HBV. The infected individuals tend to progression to liver cirrhosis and hepatocellular carcinoma. The current rHBsAg (recombinant hepatitis B surface antigen) vaccine provides protection against HBV infection, but～10%of people cannot produce neutralizing antibody after vaccination, moreover, the vaccine cannot help HBV-infected patients. Now, antiviral drugs, including the nucleoside analogous and IFN-a, used for the treatment of HBV can suppress viral replication and reduce hepatic symptoms. However, the persistence of HBV cccDNA (covalently closed circular DNA) and defective immune responses lead to treatment failure and progression to severe liver disease. Consequently, it is necessary to develop more efficient therapeutic strategies to eradicate HBV infection.HBV is unlike other enveloped viruses, which seems to avoid inducing strong innate immune responses including the type I IFN (interferon). Therefore, it may play a critical role in finding ways to induce vigorous immune responses against HBV.The unmethylated CpG (cytosine-phosphate-guanosine) motifs presented in microbial DNA can stimulate the immune system by interacting with the TLR9(pattern-recognition receptor Toll-like receptor9). Unmethylated CpG DNA can trigger immune cascades that improves antigen presentation and the secretion of cytokines, including high levels of the type I IFN. A lot of investigations indicate that CpG ODNs can provide not only a basis for improved vaccines, but also immunotherapy for infectious diseases. However, the responses to CpG ODN-based treatments were generally not sustained and was always accompanied by side effects such as toxic shock or deleterious autoimmune reactions. So far, the clinical application of CpG ODNs to HBV has not been achieved.The genome of HBV is partially double-stranded DNA, and also contains CpG motifs. Whether these HBV genome-derived CpG ODNs can induce type I IFN has not been described. In this study, we successfully identified some CpG ODNs from the HBV genome (named HBV-CpG) that are capable of inducing IFN-a production. In addition to identifying HBV-CpG, We also identified inhibitory guanosine-rich ODNs from HBV DNA (named HBV-ODNs) which have capable of inhibiting HBV-CpG-mediated IFN-α production. Nanoparticle-encapsulated HBV-CpG (termed NP(HBV-CpG)) reversed the HBV-ODN-mediated suppression of IFN-α production and activated the innate immune system. In the following study, we focused on the function of NP(HBV-CpG) in prevention and treatment of HBV infection.In our study, we designed and screened HBV-CpG and HBV-ODN by ELISA and flow cytometry. The mechanism of inhibition of HBV-ODN was analyzed by the immunofluorescence.NP(HBV-CpG) reversing the HBV-ODN-mediated-block of IFN-a production was analyzed by ELISA. ELISA and intracellular cytokine staining were used to examine the secretion of IFN-a from HBV patient-derived PBMCs. RIA and ELISA were used to detect the antibody response to HBsAg. HBV carrier mouse models were established by hydrodynamically tail intravenously injection with the pAAV/HBV1.2plasmid. ELISA and flow cytometry were used to detect the production of serum IFN-a and the activation of lymphocytes. A double-emulsion method encapsulates HBV-CpG into nanoparticles. HBsAg and anti-HBsAg antibodies were measured by RIA. The level of HBV DNA and HBcAg was tested by RT-PCR and RIA. Tissue injury was assessed by H&E staining and the measurement of ALT and bilirubin. The intracellular cytokine staining was used to examine the HBsAg-specific IFN-y-producing CD8+T cells. The major findings and conclusions of our study are shown as follows:1. HBV-CpG potently induces the production of IFN-α by human pDCs.We hypothesized that endogenous CpG ODNs from the HBV genome could interact with TLR9to induce immune responses, so an extensive screen was performed to identify the HBV-CpG in the HBV genome. Therefore, we found two candidates to potently induce IFN-α release by PBMCs. We next measured which cell type within the PBMC populations produced IFN-α in response to HBV-CpG. IFN-α was exclusively produced by Lineagel-, CD123+, HLA-DR+cell populations (pDCs) determined by flow cytometry after stimulation with HBV-CpG. So pDCs are the only cells within the PBMC populations that can response to HBV-CpG to produce IFN-α.To determine whether TLR9acted as the HBV-CpG ligand, TLR9expression was determined by flow cytometry after PBMCs were stimulated with HBV-CpG for8h. We found TLR9was significantly upregulated in PBMCs treated with HBV-CpG compared to PBMCs treated with IL-3. What is more, when we made use of chloroquine to block TLR7/9signaling, HBV-CpG-induced IFN-α production completely inhibited. We also found that there was little change in expression of TLR7on pDCs determined by flow cytometry when PBMCs were incubated with HBV-CpG.These data indicate that HBV-CpG induced a potent IFN-a response by human pDCs in a TLR9-dependent but TLR7-independent manner.2. HBV-ODN specifically blocked the HBV-CpG-mediated induction of IFN-a.Guanosine-rich ODNs specifically inhibiting TLR9signaling has been reported. Based on the considerations, we investigated HBV genome and found a high frequency of guanosine repetitive elements. We screened eight inhibitory ODNs derived from HBV genomic sequences (named HBV-ODNs) with guanosine-rich motifs. When we incubated PBMCs with HBV-CpG and different inhibitory HBV-ODNs, we found that the HBV-CpG-induced IFN-a production was inhibited. However, HBV-ODNs could not inhibit CpG-2216-induced IFN-a production. When Gen2.2cells incubated with HBV-CpG, we found that HBV-CpG colocalized with TLR9by the immunofluorescence and confocal microscopy. However, when Gen2.2incubated with HBV-CpG and HBV-ODN, HBV-ODN suppressed the uptake of HBV-CpG by Gen2.2and blocked the colocalization of HBV-CpG with TLR9.Taken together, these data suggest that inhibitory HBV-ODN specifically blocked the endogenous HBV-CpG-mediated induction of IFN-a.3. NP(HBV-CpG) reversed the HBV-ODN-mediated-block of IFN-a production.We used a double-emulsion method to encapsulate HBV-CpG into nanoparticles (termed NP(HBV-CpG)). The coadministration of PBMCs with NP(HBV-CpG) or HBV-CpG and inhibitory HBV-ODNs at a1:1ratio resulted in IFN-a production at barely detectable levels. However, the incubation of NP(HBV-CpG) and HBV-ODNs at a2:1ratio increased IFN-a production. Additional, NP(HBV-CpG) could activate NK, pDCs and T cells to a greater extent than nanoparticles and HBV-CpG. Furthermore, we also found that NP(HBV-CpG) could induce IFN-a production in HBV patient-derived PBMCs.Together, these results suggested that NP(HBV-CpG) could suppress the activity of inhibitory HBV-ODNs and stimulate strong immune responses from PBMCs. 4. NP(HBV-CpG) exerted strong immunostimulatory effects on the lymphocytes of mice.In our previous study we found that NP(HBV-CpG) could activate human PBMCs in vitro. Subsequently, we challenged wild-type mice with NP(HBV-CpG) in vivo. Mice treated intravenously with NP(HBV-CpG) displayed higher levels of CD40and CD80on pDCs and cDCs than mice treated with PBS. Meanwhile, the expression of ICOS and CD69was strongly upregulated on the NK and T cells treated with NP(HBV-CpG) compared to the PBS treated. Furthermore, the coincubation of splenic lymphocytes with NP(HBV-CpG) increased the expression of CD80and CD40on pDC and cDC but also the expression of CD69on NK and T cells in agreement with the in vivo experiment. NP(HBV-CpG) treated also increased the viability of the pDCs and cDCs.These experiments indicate that NP(HBV-CpG) can induce a strong immunostimulatory effect on murine lymphocytes in vitro and in vivo.5. NP (HBV-CpG) enhanced the antibody response to HBsAg.BALB/c and C57BL/6mice were divided into four groups, including the rHBsAg vaccine plus NP(HBV-CpG), the rHBsAg vaccine plus NPs, rHBsAg vaccine alone and untreated control. The level of anti-HBsAg antibodies in the sera of the mice was examined2and4weeks after the final immunization. Immunization with the rHBsAg vaccine plus NP(HBV-CpG) greatly increased the anti-HBsAg antibody levels in both mice. Significantly, NP(HBV-CpG) administration was a large increase in the production of Thl-dependent IgG2a-antibodies.Together, these results suggested that NP(HBV-CpG) as an adjuvant can assist the rHBsAg vaccine in inducing vigorous Thl-biased anti-HBsAg antibody responses.6. NP (HBV-CpG) stimulated robust innate immune responses in HBV carrier mice.Given that NP(HBV-CpG) exerted a strong immunostimulatory effect on wild-type mice, we evaluated the effect of NP(HBV-CpG) on HBV carrier mice. The mouse model established by hydrodynamically tail intravenously injection with the pAAV/HBV1.2plasmid. The HBV carrier mice were administered NP(HBV-CpG), NPs, HBV-CpG or PBS respectively. The NP(HBV-CpG) treatment increased the expression of CD40and CD80on cDCs, ICOS and CD69on NK cells and CD69on CD4+and CD8+T cells. Significantly, high levels of serum IFN-α, but not IFN-β, were observed in the NP(HBV-CpG) treatment.These results indicated that NP(HBV-CpG) can trigger a robust immune response in HBV carrier mice.7. NP(HBV-CpG)-based theatment effectively cleared HBV in HBV carrier mice.Previous study had reported that the HBV carrier mice exhibit tolerance toward HBsAg because HBsAg failed to induce an immune response in these mice. However, our study indicated that NP(HBV-CpG) could stimulate the activation of lymphocyte, especially IFN-α expression in HBV carrier mice. Therefore, we speculated NP(HBV-CpG) may have the effect on the clearance of HBV. In fact, after we treated HBV carrier mice with NP(HBV-CpG) combined with rHBsAg or NPs combined with rHBsAg, we found that the HBsAg level in the serum of carrier mice treated with NP(HBV-CpG)-baesd treatment declined significantly. Actually HBV surface antigenemia disappeared in about90%of carrier mice within the NP(HBV-CpG)-based trerapy. However, the HBsAg level remained high in the mice treated with rHBsAg combined with NPs. Moreover, the HBcAg level in the liver tissues and the HBV DNA level in the serum declined significantly and were undetectable in some of the samples receiving the NP(HBV-CpG)-based treatment. Furthermore, anti-HBsAg antibodies began to appear after HBsAg clearance in the groups treated with NP(HBV-CpG) combined with rHBsAg. Moreover, the percentage of HBsAg-specific IFN-y-producing CD8+T cells upregulated by the NP(HBV-CpG)-based therapy that suggest that the combination treatment augmented CTL function. Meanwhile, the livers of the HBV carrier mice receiving the NP(HBV-CpG) combined administration showed normal architecture with mild inflammatory infiltrate. Normalization of serum bilirubin and ALT further suggested that HBV carrier mice had good tolerance to the NP(HBV-CpG)-based treatment.Taken together, these data suggest that NP(HBV-CpG)-based theatment can effectively eradicates HBV infection, thereby exerting strong anti-HBV activity.更多还原