摘要
研究目的
乙肝病毒(hepatitis B virus, HBV)感染可以造成急慢性肝脏疾病,如病毒性肝炎、肝硬化及肝癌等,是危害人类健康的主要病因。我国HBV携带者约占全国总人口的10%,是乙型肝炎的高发区。目前临床使用抗HBV感染的典型药物有拉米夫定和α-干扰素,但是存在容易诱发病毒耐药突变以及副作用较多的问题,限制了疗效的发挥。HBV感染能够破坏机体免疫平衡,导致机体抗病毒免疫功能低下或紊乱,因此,抑制HBV感染需要激活机体抗病毒免疫应答。给易感人群接种乙肝疫苗是预防HBV感染的有效手段,但是仍有5%-10%的人接种后抗体水平很低或者无应答。铝佐剂是目前唯一可以用于乙肝疫苗的佐剂,虽然较安全,但是因其不能引起机体细胞免疫应答,使疫苗在预防和治疗HBV感染方面存在一定的不足,因而研制新型乙肝疫苗佐剂尤为重要。
可德兰多糖(curdlan)是由产碱杆菌(Alcaligenes faecalis)发酵产生的一种胞外多糖,具有免疫调节、抗肿瘤、抗炎、抗凝血等活性,因其安全无毒且加热可成凝胶,被FDA批准用于食品工业。研究发现,curdlan硫酸化产物—硫酸化可德兰(curdlan sulfate)具有良好的抗HIV活性,并已对其开展Ⅱ期临床试验。鉴于curdlan和curdlan sulfate二者良好的免疫调节及抗病毒活性,我们期望通过本课题研究能够得到一种具有抗病毒感染作用的免疫调节剂,能够在免疫治疗方面发挥抗HBV感染的作用,同时还能增强重组乙肝表面抗原(hepatitis B surface antigen, HBsAg)的免疫原性,改善疫苗在预防和治疗HBV感染方面的应用。
通过研究curdlan sulfate对小鼠免疫相关细胞的影响,考察其体外免疫调节活性,采用表面等离子共振(surface plasmon resonance, SPR)技术研究其结合的受体；利用HepG2.2.15细胞上清液浓缩得到的HBV感染HepG2及HepaRG细胞,研究curdlan sulfate是否可以干扰病毒感染细胞的过程,并用SPR技术研究多糖与重组HBsAg的结合情况；最后,将curdlan sulfate与重组HBsAg联合注射BALB/c小鼠,研究其能否增强疫苗的免疫原性,提高产生的抗体水平。本课题研究将为curdlan sulfate开发成为一种新型抗HBV的免疫预防及治疗剂提供重要的依据和基础。
研究方法
1Curdlan sulfate的制备及结构表征
首先我们采用DMSO为溶剂、SO3-吡啶为硫酸化试剂,对curdlan进行修饰。通过控制反应温度,制备出四种curdlan sulfates。采用红外光谱分析技术鉴定所制备样品结构,凝胶渗透色谱-多角度激光散射(GPC-MALLS)联用技术测定重均分子量(Mw),元素分析仪测定硫(S)含量。
2Curdlan sulfate体外免疫调节活性及机制的研究
分离小鼠脾淋巴细胞,采用MTT法测定curdlan sulfate对其增殖的影响。采用小鼠RAW264.7细胞系,研究curdlan sulfate对巨噬细胞活化的作用,包括：用细胞吞噬中性红和FITC-dextran实验来分析其对巨噬细胞吞噬功能的影响；Griess试剂法测定RAW264.7细胞经curdlan sulfate作用后产生NO的水平；ELISA试剂盒测定curdlan sulfate对RAW264.7分泌IL-6、IL-1β及TNF-α细胞因子的影响；iNOS、IL-6、IL-1β和TNF-a的mRNA水平采用荧光定量PCR (FQ-PCR)进行分析。分离并培养小鼠骨髓来源树突状细胞(BMDCs),采用流式细胞术检测curdlan sulfate对BMDCs表面成熟标志分子表达的影响。机制研究采用Westernblot实验检测RAW264.7细胞中丝裂原活化蛋白激酶(MAPKs)途径相关蛋白的表达以及NF-κB的活化情况,并运用SPR技术分析curdlan sulfates与小鼠重组dectin-1的结合情况。
3Curdlan sulfate体外抗HBV感染活性及机制的研究
采用PEG8000对HepG2.2.15细胞上清液进行浓缩得到HBV,用其感染HepG2及HepaRG细胞。采用FQ-PCR测定受感染细胞中HBV DNA的含量来考察curdlan sulfate对HBV黏附及入胞过程的影响；用HBsAg及HBeAg ELISA试剂盒检测受感染细胞培养第7d的细胞上清中HBsAg及HBeAg的含量,来考察curdlan sulfate对HBV感染细胞的影响；采用氯酸钠及硫酸乙酰肝素酶(HPA)处理HepG2及HepaRG细胞,用FQ-PCR检测HBV感染细胞的情况,并进一步用SPR技术分析curdlan sulfates与重组HBsAg的结合情况,探讨硫酸根在curdlansulfate与HBsAg结合中的作用,并进一步分析其作用机制。
4Curdlan sulfate体内用作乙肝疫苗佐剂的研究
Curdlan sulfate与重组HBsAg联合免疫BALB/c小鼠,于第0、14、28d共接种3次,第35d取小鼠脾淋巴细胞,采用流式细胞仪检测脾淋巴细胞巨噬细胞募集和树突状细胞成熟、CD4+、CD8+分群以及CD4活化情况；取小鼠脾淋巴细胞进行体外培养并用HBsAg再刺激,测定其增殖能力以及分泌IFN-γ、IL-17A、 IL-4、IL-10的水平；流式细胞术胞内染色法分析小鼠脾淋巴细胞中Th1、Th2及Th17类细胞因子的胞内表达情况。另外,在每次免疫后7d收集小鼠血清,用anti-HBs ELISA试剂盒测定血清中抗体的含量,并用间接ELISA方法测定anti-HBs免疫球蛋白亚型IgG1、IgG2a的含量,分析小鼠产生的免疫应答类型。
研究结果
1Curdlan sulfate的制备及结构表征
通过控制反应条件,共制备了四种不同分子量及S含量的curdlan sulfates,分别命名为CS1、CS2、CS3及CSⅢ。红外光谱检测结果显示,curdlan sulfates在curdlan原有特征吸收峰基础上,在1258cm-1及816cm-1附近处出现了不对称S=O的伸缩振动峰及对称C-O-S的伸缩振动峰,说明curdlan已被成功硫酸化修饰。CS1、CS2、CS3和CSIII的Mw经GPC-MALLS测定为25.87kDa、87.03kDa、171.9kDa和62.54kDa,S含量分别为(4.806±0.30)%、(6.288±0.03)%、(9.340±0.16)%和(7.312±0.11)%。由于CS3含有最高的S含量,下面的活性检测均采用CS3样品进行实验。
2CS3体外免疫调节活性与MAPKs途径有关
CS3促进小鼠细胞脾淋巴细胞增殖效果显著,在400μg/mL时增殖率达到85.87%,但对RAW264.7细胞的增殖没有明显作用。CS3能增强RAW264.7吞噬中性红及FITC-dextran的能力,显著提高细胞产生NO、IL-6、IL-1β及TNF-α的水平,而且对细胞中iNOS、IL-6、IL-1β和TNF-a mRNA的水平也有一定的提高,并呈剂量依赖性。另外,CS3可促进BMDCs上调表达MHCⅡ、CDllc、 CD40、CD80、CD86分子,产生IL-12和IL-6,刺激细胞成熟。
Western blot结果显示CS3可以通过促进MAPKs途径中Erkl/2和SAPK/JNK的磷酸化来传递信号,活化NF-κB,提高iNOS的表达。SPR技术表明curdlan sulfates可以与curdlan已知受体dectin-1结合,说明curdlan经硫酸化修饰后主要受体并未改变。
3CS3可以抑制HBV感染的黏附过程
FQ-PCR结果显示,CS3可以抑制HBV与HepG2及HepaRG细胞的黏附,500μg/mL时的抑制率为分别52.06%、50.43%；HBV感染细胞后上清中HBsAg及HBeAg的测定结果表明,CS3可以抑制HBV对细胞的感染。CS3对黏附过程的抑制率远高于其对HBV感染细胞的抑制率,说明CS3抗HBV感染的作用阶段主要是干扰病毒黏附入胞过程,而不是干扰病毒DNA复制等过程。
氯酸钠和HPA作用的细胞不表达或低表达硫酸乙酰肝素(HS),用HBV感染分别经氯酸钠和HPA处理后的HepG2细胞,发现病毒感染水平分别降低为阴性对照的(11.44±2.30)%和(57.17±0.79)%；感染经二者处理后的HepaRG细胞,感染率分别降低为阴性对照的(9.86±2.29)%和(49.70±±16.07)%。SPR技术分析发现curdlan sulfates可以与重组HBsAg结合,并且硫酸根含量越高,亲和力越强,提示CS3可能与HBV包膜蛋白结合,干扰HBV入胞。
4CS3可以增强重组HBsAg免疫原性
流式细胞术检测经3次免疫接种后的小鼠脾淋巴细胞结果表明,CS3联合重组HBsAg免疫小鼠,可以促进巨噬细胞在脾脏中的募集,F4/80阳性细胞数增加,占脾淋巴细胞总数的(34.27±±3.47)%,明显高于商品HB疫苗(HB vaccine)组(**p<0.01)；同时可以刺激树突状细胞的成熟,使细胞高表达MHCⅡ、CD11c、CD40和CD86分子；CD4+及CD8+T细胞亚群在脾淋巴细胞中的比例明显升高,与HB vaccine相比有显著性差异(*p<0.05),且CD4+CD69+细胞数与单独注射HBsAg相比有显著性增加；分离小鼠脾淋巴细胞进行HBsAg体外再刺激,发现经CS3作用的小鼠脾细胞增殖能力增强,分泌Th1类型细胞因子IFN-y的量明显高于HB vaccine组(**p<0.01),CS3在高剂量时可以促进Th2类型细胞因子IL-4和IL-10的产生,但在低、中剂量时则作用不明显甚至有一定的抑制作用,而对Thl7类细胞因子IL-17A则没有明显作用；采用流式细胞术分析胞内Thl及Th2类细胞因子蛋白的表达情况与细胞上清中测定结果一致。
小鼠血清中anti-HBs检测结果显示,CS3可以明显提高HBsAg产生的anti-HBs水平,高剂量组要明显优于HB vaccine组。Anti-HBs免疫球蛋白亚型IgGl的水平经CS3作用后下降,IgG2a的水平显著升高,且Ig2a/IgG1的值与HB vaccine组相比明显升高(*p<0.05),说明CS3用作HBsAg佐剂可以使机体免疫类型向Thl方向发展。创新性和结论
(1)首次采用DMSO/SO3吡啶反应体系,制备了不同Mw和S含量的curdlan sulfates,此法安全方便,毒性较低,并且所得样品的Mw和S含量随反应温度的升高而降低。
(2)首次研究了curdlan sulfate的体外免疫调节活性,样品CS3可以促进小鼠脾淋巴细胞增殖、RAW264.7细胞活化及BMDCs成熟。
(3)首次采用SPR技术研究了curdlan sulfate与curdlan已知受体dectin-1的结合情况,确定dectin-1是curdlan sulfate的主要受体,明确CS3发挥体外免疫调节活性的机制是通过与免疫细胞表面dectin-1受体结合,激活MAPKs信号通过中的Erkl/2和SAPK/JNK途径,导致NF-κB向核内移位,调节相关基因的转录。
(4)首次研究了curdlan sulfate体外抗HBV感染的活性,发现CS3可以抑制HBV感染HepG2和HepaRG细胞,并且主要是干扰HBV黏附细胞的过程。
(5)采用氯酸钠、HPA处理HepG2和HepaRG细胞发现HS在HBV感染细胞过程中发挥一定的作用,借助SPR技术研究curdlan sulfates与重组HBsAg的结合能力,提示CS3发挥体外抗HBV感染活性的机制可能是通过与HBV包膜蛋白结合,干扰HS-HBV的相互作用,影响HBV与靶细胞的黏附和融合。
(6)首次进行了curdlan sulfate用作乙肝疫苗佐剂的研究,CS3可以提高重组HBsAg的免疫原性,增强小鼠机体的免疫水平,提高血清中抗体滴度,并且主要诱导机体免疫应答向Thl型发展。
Objectives:
The infection of hepatitis B virus (HBV) can cause acute and chronic liver diseases including viral hepatitis, cirrhosis and hepatocellular carcinoma (HCC), so it is a leading risk threatening the public health. China is the region of high hepatitis B (HB) incidence, where the rate of HBV carriers in the general population is about10%. Currently, the typical anti-HBV infection drugs used in clinic are lamivudine and interferon (IFN)-a, but they are likely to cause drug-resistant HBV mutants and some adverse effects, which limit their anti-HBV effect. The HBV infection can destroy the balance of body's immune system, leading to the defect or disorder of host anti-viral immunity. Therefore, the activation of host anti-viral immune is essential for inhibiting HBV infection. One effective way to prevent susceptible people from virus infection is to immunize them with HB vaccine. However, nearly5%-10%of immunized individuals do not respond adequately to the vaccine and remain at risk for HB infection. At present, alum (aluminum hydroxide) remains the only adjuvant for HB vaccine approved for human use, although it is safe, but unfortunately, it has no effect on cellular immunity, which makes the HB vaccine lack of enough preventive and therapeutic effect. Therefore, novel adjuvants are urgent to be developed.
Curdlan is an extracellular polysaccharide produced by the bacteria of Alcaligenes faecalis, possessing immunomodulatory, anti-tumor, anti-inflammatory, anti-coagulant activities, and so on. Since curdlan is safe and has gel-forming property when heated, it has been approved by FDA to be used in food industry. Curdlan sulfate, prepared by curdlan sulfation modification, has been found to own excellent anti-HIV activity, and curdlan sulfate used as an anti-HIV drug has been under going phase II clinical study. Considering curdlan possesses favorable immunomodulatory effect, while curdlan sulfate has good anti-viral activity, we expect to obtain an immunomodulator owning anti-viral potency, which will play a part in immunotherapy of HBV infection, as well as enhancing recombinant hepatitis B surface antigen (HBsAg) immunogenicity to improve the applications of vaccine in HB prevention and therapy.
We investigated the in vitro immunomodulatory activity of curdlan sulfate through studying its effect on immune cells. Surface plasmon resonance (SPR) technology was used to study the binding receptor of curdlan sulfate. The HBV particles was prepared from concentrated culture supernatant of HepG2.2.15cells, and were used to infect HepG2and HepaRG cells to find out whether curdlan sulfate interfered with HBV infection process. The binding affinity of curdlan sulfate with recombinant HBsAg was also analyzed by SPR technology. In order to study whether curdlan sulfate could be used as an adjuvant, we combined curdlan sulfate with recombinant HBsAg to immunize BALB/c mice and analized the anti-HBs level in the immunized mice. Our research may provide critical basis for developing curdlan sulfate as a novel immunoprophylaxis and immunotherapy agent.
Methods:
1. Preparation and characterization of curdlan sulfates
We first prepared four kinds of curdlan sulfates by modification of curdlan with DMSO/sulfur trioxide-pyridine reaction system, through controlling the reaction time. Then, the structure of the samples were characterized by FT-IR spectroscopy, the weight-average molecular weight (Mw) and sulfur (S) content were determined by GPC-MALLS (gel permeation chromatography-multi angel laser light scattering) and element analysis.
2. In vitro immunomodulatory activity and mechanism study of curdlan sulfate
MTT method was used to determine the proliferative effect of curdlan sulfate on mouse spleen lymphocytes. The effect of curdlan sulfate on RAW264.7cell activation was evaluated as follows:the phagocytosis function was studied by cell phagocytosis of neutral red and FITC-dextran assays; the level of NO produced by cells was determined with Griess regent method; the secretions of IL-6, IL-1β and TNF-a cytokines were assayed with ELISA kits; and the mRNA levels of iNOS, IL-6, IL-1(3and TNF-a were determined with fluorescence quantitative (FQ)-PCR technology. To investigate the effect of curdlan sulfate on dendritic cells (DCs) maturation, we prepared and cultured mouse bone marrow dendritic cells (BMDCs), and used flow cytometry to detect the cell surface maturation markers.
The action mechanism was studied through analyzing the mitogen-activated protein kinases (MAPKs) signaling pathway related proteins as well as NF-κB expression by Western blot assay. The binding affinity of curdlan sulfates with mouse recombinant dectin-1was also studied with SPR technology.
3. In vitro anti-HBV activity and mechanism study of curdlan sulfate
The HBV used to infect HepG2and HepaRG cells was prepared by concentration of the culture supernatant of HepG2.2.15cells by PEG8000. The effect of curdlan sulfate on HBV attachment process were evaluated by detecting HBV DN A content in infected cells by FQ-PCR, while the effect of curdlan sulfate on HBV infection was analyzed by determining the HBsAg and HBeAg content in the supernatant of cells on day7after infection by ELISA kits.
For the mechanism study, we first treated the HepG2and HepaRG cells with sodium chlorate or heparanase (HPA), and then determined the HBV DNA content in infected cells by FQ-PCR. We also studied the binding affinity of curdlan sulfate with recombinant HBsAg, to find out what role of sulfate groups play in the binding interaction.
4. In vivo study of curdlan sulfate acting as an HB vaccine adjuvant
Curdlan sulfate and recombinant HBsAg were used together to inoculate BALB/c mice for three times on day0,14,28, respectively. On day35, the mouse splenocytes were prepared and analyzed by flow cytometry for studying the recruitment of macrophages, activation of DCs, CD4+, CD8+T-cell subpopulations and activation of CD4+cells. The splenocytes restimulated with HBsAg in vitro were used to test the cell proliferative ability and the secretion of IFN-y, IL-17A, IL-4, IL-10cytokines. The expressions of intracellular Thl, Th2and Th17-type cytokines were analyzed by flow cytometry with intracellular staining method. In addition, the titer of anti-HBs in mouse serum collected at the7th day after each immunization was determined by anti-HBs EIA kit. The HBsAg-specific IgGl and IgG2a antibodies in serum were detected by an indirect ELISA, in order to analyze the type of immune response.
Results:
1. Preparation and characterization of curdlan sulfate
Through controlling the reaction condition, four kinds of curdlan sulfates with different Mw and S content, were prepared and named as CS1, CS2, CS3, and CSIII, respectively. The results of FT-IR spectra indicated that in addition to the characteristic absorption peaks of curdlan, curdlan sulfates also showed new characteristic absorption bands near1258cm"1and816cm'1, which represent asymmetrical S=O stretching vibration and symmetrical C-O-S stretching vibration. This result confirmed that sulfate groups had been incorporated into the curdlan backbone. The Mws of CS1, CS2, CS3and CSIII determined by GPC-MALLS were25.87,87.03,171.9and62.54kDa, and the S contents detected by element analysis were (4.806±0.30),(6.288±0.03),(9.340±0.16) and (7.312±0.11)%, respectively. We chose CS3as the study object in the following activity studies as it had the highest S content among the prepared compounds.
2. In vitro immunomodulatory activity of CS3was correlated to MAPKs signaling pathway
CS3promoted the proliferation of mouse splenocytes significantly, and the proliferation rate reached85.87%at the concentration of400μg/mL. However, it had no apparent effect on the proliferation of RAW264.7cells. CS3could greatly enhance the ability of RAW264.7cell phagocytosis of neutral red and FITC-dextran, increase the production of NO, IL-6, IL-1β and TNF-α, as well as the mRNA levels of iNOS, IL-6, IL-1β and TNF-α, in a dose-dependent manner. Besides, CS3stimulated the maturation of BMDCs by the cell performance of up-regulating MHCII, CD11c, CD40, CD80, CD86, and production of IL-12and IL-6. The results of Western blot showed that CS3could activate MAPKs pathway through Erkl/2, SAPK/JNK signalings, and further induce the translocation of NF-κB into nuclei, where it regulate the iNOS gene transcription. The SPR technology indicated that curdlan sulfates could bind with the curdlan receptor, dectin-1, and the sulfation modification of curdlan did not change its primary receptor.
3. CS3could inhibit the attachment process of HBV infection
The FQ-PCR results demonstrated that CS3could inhibit the attachment of HBV to HepG2and HepaRG cells, and the inhibition rate was52.06%and50.43%at500μg/mL, respectively. The detection of HBsAg and HBeAg in the cultured supernatant indicated that CS3could also depress HBV infecting the cells. That the inhibition rate of attachment was much higher than that of the whole infection process revealed that CS3mainly interfered with the attachment, not HBV DNA replication steps, and other else.
The cells treated with sodium chlorate or HPA express low or no heparan sulfate (HS). HepG2cells treated with sodium chlorate or HPA separately were infected with HBV, and the results showed that the infection rates were reduced to (11.44±2.30)%and (57.17±0.79)%of the negative control, respectively. HepaRG cells treated with sodium chlorate or HPA separately were infected with HBV, and the results displayed that the infection rates were reduced to (9.86±2.29)%and (49.70±16.07)%of the negative control. SPR analysis found that curdlan sulfates could bind with recombinant HBsAg, and the higher content of the sulfate groups the stronger of the binding affinity, indicating that CS3may bind with HBV envelope proteins, and interfere HBV entry into host cells.
4. CS3could enhance recombinant HBsAg immunogenicity
The flow cytometry analysis of splenocytes from mice after three immunizations, suggested that CS3could enhance the immune response of mice induced by recombinant HBsAg. CS3increased the recruitment of macrophages with F4/80positive cells accounted for (34.27±3.47)%of the total splenocytes, which was notably higher than that of commercial HB vaccine group (**p<0.01). The expressions of MHCII, CD11c, CD40and CD86were upregulated by CS3, indicating that CS3could stimulate the maturation of DCs. The percentages of CD4+and CD8+T cells in CS3group were increased significantly, compared with HB vaccine group (*p<0.05). Also, the number of CD4+CD69+T cells was much more in mice immunized with CS3and HBsAg than that with HBsAg alone. The splenocytes proliferation in the mice immunized with CS3was significantly higher than that in the HBsAg and HB vaccine groups, and the production of Thl-type cytokine IFN-y was increased greatly, compared with HB vaccine group (**p<0.01). However, CS3could only increased the production of Th2-type cytokines IL-4and IL-10obviously at high dosage, while had no apparent or even inhibition effect at low and middle dosages. Besides, CS3seemed to have no effect on the production of Th17-type cytokine IL-17A. The intracellular Thl and Th2-type cytokines expression in mice splenocytes showed the similar results to those tested in the cell culture supernatant.
The anti-HBs titer in mice sera was raised significantly in CS3group, and at high dosage, the effect was much better than that of HB vaccine group. The level of anti-HBs specific IgGl subclass was decreased induced by CS3, while IgG2a was increased, and the ratio of IgG2a/IgGl was also raised obviously, compared with HB vaccine group (*p<0.05), which indicated that CS3acting as the HBsAg vaccine could induce the immune response biased towards Thl type.
Novelties and Conclusions:
(1) The preparation of curdlan sulfates with different Mw and S content through the reaction of DMSO/SO3-pyridine system was firstly achieved. This reaction method was safe, convenient and low toxic. The Mw and S contents of curdlan sulfates were closely related to the reaction temperature, the lower the reaction temperature, the higher Mw and S content of curdlan sulfates.
(2) The in vitro immunomodulatory activity of curdlan sulfate was first studied. CS3could enhance the proliferation of mouse splenocytes, activation of RAW264.7cells, and maturation of BMDCs.
(3) SPR technology was used to analyze the binding affinity of curdlan sulfate with curdlan receptor-dectin-1for the first time, and it was confirmed that dectin-1was the primary receptor for curdlan sulfates. The mechanism of CS3acting as an immunomodulator in vitro could be elucidated that CS3could bind with the receptor dectin-1expressed on the immune related cells, and further activate MAPKs through Erkl/2and SAPK/JNK signalings, which lead to NF-κB translocation to nuclei and regulate some gene transcriptions.
(4) The anti-HBV infection effect of curdlan sulfate was first studied in vitro. The results showed that CS3could inhibit HBV infection of HepG2and HepaRG cells mainly through interfering with the attachment process.
(5) The treatment of HepG2and HepaRG cells by sodium chlorate or HPA showed that HS played a role in HBV infecting the cells. SPR technology analysis showed that curdlan sulfates could bind with the recombinant HBsAg, which suggested that CS3may bind with HBV envelope proteins, interfere with HS-HBV interaction, and influence the attachment onto and entry into the host cells.
(6) Curdlan sulfate acting as an adjuvant of recombinant HBsAg was evaluated for the first time. CS3could enhance the immunogenicity of HBsAg, increase the immune response of mice, boost anti-HBs titer in mouse sera, and induce the immune response biased towards Thl type.
引文
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