lifevendor 发表于 2011-5-30 12:34
Therapeutic vaccines and immune-basedtherapies for thetreatment of chronichepatitis B: perspectives ...
Therapeutic vaccines and immune-basedtherapies for the
treatment of chronichepatitis B: perspectives and challenges
慢性乙肝的治疗性疫苗和基于免疫的治疗方案：
前瞻和挑战
Marie-Louise Michel 1，2，3, Qiang Deng 4 andMaryline Mancini-bourgine1,2
1 Institut Pasteur, Laboratoire de Pathogenèsedes virus de l'hépatite B, département
de virologie ; 75015 Paris, France
2 INSERM U845, Centre de recherche «Croissance et signalisation », Faculté de
Médecine Paris Descartes ; 75015 Paris,France
3 AP-HP, Paris, France
4 Institut Pasteur of Shanghai, Unit ofTumor Virology, 200025 Shanghai, China.
缩写：HBV： Hepatis B virus 乙肝病毒
CccDNA：covalently closedcircular DNA 共价闭合环状DNA
HbeAg： hepatitis B ‘e’ antigen乙肝e抗原
HbsAg：hepatitis B surfaceantigen 乙肝表面抗原
NK,natural killer; 自然杀伤细胞
IFN,interferon; 干扰素
IL,interleukin; 白细胞间介素
TNF,tumor necrosis factor; 肿瘤坏死因子
DCs,dendriticcells; 树突状细胞
PD-1,programmeddeath-1; 程序死亡-1
PD-L1programmeddeath-ligand 1; 程序死亡-配体1
CTLA-4,cytotoxic T lymphocyte antigen 4; 细胞毒性T淋巴细胞抗原-4
Bim,Bcl2-interacting mediator of cell death; 互动调节
Tregs,regulatoryT cells; 调控性T细胞
mDCs,myeloid DC; 骨髓的树突状细胞
pDCs, plasmacytoid DCs; 类浆树突状细胞
IC,immunecomplexes; 免疫复合体
HBcAg,hepatitisB core antigen; 乙肝核心抗原
Th1,T helper 1; T辅助细胞
PBMCs,peripheral blood mononuclear cells; 外周血单核细胞
CIK,cytokine-inducedkiller; 细胞因子导致的杀伤细胞
TCR,T-cell receptor; T-细胞受体
ALT,alanine aminotransferase 转氨酶
致谢：翻译这篇文章的资金支持来自Stephen Wong。
摘要
Thetreatment of chronic hepatitis B virus (HBV) infection has greatly improvedover the last
10years, but alternative treatments are still needed. Therapeutic vaccination isa promising
newstrategy for controlling chronic infection. However, this approach has not beenas
successfulas initially anticipated for chronic hepatitis B. General impairment of theimmune
responsesgenerated during persistent HBV infection, with exhausted T cells notresponding
correctlyto therapeutic vaccination, is probably responsible for the poor clinicalresponses
observedto date. Intensive research efforts are now focusing on increasing the efficacyof
therapeuticvaccination without causing liver disease. We describe here new approaches, for
usewith therapeutic vaccination, to overcome the inhibitory mechanisms impairingimmune
responses.We also describe innovative strategies for generating functional immuneresponses
and inducing sustained control of thispersistent infection.
乙肝的治疗在过去的10年里有了很大的改善，但是仍然需要有的新的治疗方案。治疗性疫苗是一个新的非常有前景的
控制慢性感染的策略，但是，对慢性乙肝该方案并不如同最初的期望那样成功。通常，长期的乙肝感染可以导致免疫
反应的损伤，耗竭的T细胞并不准确的对治疗性疫苗响应，这可能可以解释目前观察到的差的响应。目前，大部分研
究的努力都聚集在提高治疗性疫苗的有效率，但同时避免引起肝部疾病。我们在这里描述一种新的途径，使用治疗性
疫苗，从而来克服破坏免疫系统的抑制机制（inhibitory mechanisms）。我们同时描述了一项新的策略用于产生功
能性的免疫反应并可以导致持续性的控制残留顽固的感染。
Worldwide,two billion people have been infected with the hepatitis B virus (HBV) at
sometime, more than 370 million currently have chronic HBV infection, and about one
milliondie each year from HBV-related liver disease. HBV is a non cytopathic virus,and the
hostimmune response determines whether the virus is cleared or whether immunopathyand
liverdamage are induced. Persistent inflammation during chronic HBV infectionresults in
livercirrhosis or hepatocellular carcinoma in 25% of patients. HBV is currently thesecond
leadingcarcinogen after tobacco, with up to 80% of hepatocellular carcinoma cases
worldwideattributable to HBV [1]. Individuals with chronic HBV infection also serve asthe
primaryreservoir for viral spread. Preventive vaccination is the most effective way toreduce
theglobal incidence of hepatitis. Efficient vaccines against hepatitis B have beenavailable for
over20 years, and their use should decrease the incidence of HBV infection [2]. Bycontrast,
thetreatment of chronic HBV infection is based on the use of antiviral agents.These agents
haveimproved considerably over the last 10 years, with the development of molecules
targetingthe HBV polymerase and decreasing viral replication. The major goals ofanti-HBV
treatmentare to prevent the development of progressive disease, specifically cirrhosis,liver
failureand hepatocellular carcinoma. However, although currently available antiviraldrugs
efficientlydecrease serum viral load to undetectable levels, they fail to eradicateinfection due
tothe persistence of HBV covalently closed circular DNA (cccDNA) in hepatocytesand the
emergenceof resistant viruses [3, 4]. In addition, such drugs rarely result in thelong-term
immunologicalcontrol of HBV infection through the elimination of residual infected
hepatocytes,hepatitis B “e” antigen (HBeAg) seroconversion and/or hepatitis B surface
antigen(HBsAg) clearance. Moreover, long-term treatment is expensive and may result in
problems of toxicity andintolerance.
全世界，有2亿人被乙型肝炎病毒感染，超过370百万目前受到慢性乙肝感染，每年有100万死于HBV相关的肝部疾病。
HBV是一个不引起细胞病变的病毒，体内的免疫反应来确定病毒是否清楚或者是免疫性疾病和导致肝脏损伤。
在慢性感染者中，由于持续的炎症导致25%的患者转向肝纤维化或者是肝细胞癌化。HBV病毒是仅次于烟草的
第二大致癌因素，全世界超过80%的肝癌是由于HBV引起【1】。慢性乙肝病毒感染者是乙肝病毒传播的主要来
源预防性的疫苗接种是最有效的降低全球乙肝发生率方式。针对乙肝的有效的疫苗已经出现了20年，疫苗的广泛使
用降低了HBV的感染【2】。另一方面，治疗慢性乙肝感染主要基于抗病毒的药物，过去的10年，这些药剂有了很大
的改进，针对DNA聚合酶的分子有效的降低了病毒的复制，抗-HBV的主要治疗是为了阻止病情的持续发展，
尤其是肝硬化，肝衰竭和肝癌。但是，尽管现有的药物可以将血清中的病毒含量下降到检测范围以下，他们却不能
完全清除HBV，因为在肝细胞中有cccDNA的存在和耐药病毒的出现【3,4】。此外，这些药物很少能够达到长期的
通过清楚残余的感染肝细胞来免疫控制HBV的感染，比如说HbeAg血清转换或者是HbsAg的清除。更多的是，长期的
服药是昂贵的并且可能导致药物毒性和耐受问题（intolerance）。
Boththe adaptive and innate immune responses are known to be involved in viral
clearanceduring HBV infection [5]. There is a clear dichotomy in the profile of theimmune
responsesobserved, depending on whether patients naturally resolve viral infection or
developchronic infection. Patients with self-limited acute hepatitis B displaymulti-specific
CD4and CD8 T-cell responses, with the secretion of antiviral cytokines and theproduction of
anti-HBVantibodies. By contrast, patients suffering from chronic infection have veryweak or
functionallyimpaired immune responses. Therapeutic vaccination has been proposed as a
potentiallypromising strategy for controlling viral infection, based on these observations.
Therapeuticvaccination aims to eliminate persistent viral infection, by stimulating the
patient’simmune responses.
我们知道在HBV感染中，先天和后天适应性（adaptive）的免疫反应参与到了病毒的清除中【5】。
在免疫响应中可以发现两种清晰的不同的信息（profile），取决于病人是否自发的清除病毒感染或者是发展成慢性感染。
患者带有自限制（self-limited：指不通过药物治疗，体内自己治愈）自治愈的急性感染显示多重特异性的CD4和CD8 T细胞响应，有分泌抗病毒的细胞
因子和产生抗病毒的抗体。相反，慢性感染患者有非常弱的或者功能损坏的免疫响应。基于以上这些观察，治疗
性疫苗刚开始被认为是非常有潜力的用于控制病毒的感染的治疗方案。治疗性疫苗瞄准的是通过激发病人的免疫
反应来清除长期持续的病毒感染。（ps：self-limited:不经治疗自己可以治愈的）
In this review, we firstoutline the characteristics of cell-mediated immune responses
andthe immunosuppressive environment generated during chronic HBV infection. Wethen
describecurrent and future approaches for manipulation of the immune system to achieve
sustainedcontrol of this persistent infection.
在这篇综述中，我们首先描述由细胞调节的免疫响应的特点和由慢性乙肝感染导致的免疫耐受
or免疫抑制？（immunosuppressive）环境。我们然后将描述当前和将来有哪些途径可以用来通过
调节免疫系统来达到持续的控制这个顽固的感染。
The immune response to viral infection isorchestrated in several stages, which function together to eliminate thepathogen and leave the host with memory cells for defense against subsequentinfections. During the early phases of acute HBV infection, natural killer (NK)cells are the first line of host defense, and the activation of these cellshelps to reduce viral load, through the secretion of interferon (IFN)-γ [6].However, the activation of these cells is rapidly checked and this wastemporally associated with a surge in interleukin (IL)-10 at the time of peakviremia [7]. Adaptive immunity is then induced by 5-6 weeks postinfection, theCD8 and CD4 T-cell populations expand and become major contributors to viral control.HBV-specific T-cell responses participate in the elimination of infectedhepatocytes, initially through antiviral cytokine secretion (IFN-γ and tumornecrosis factor (TNF)-α) and then through the production of cytotoxicmolecules, once the level of MHC-class I peptide complexes on infected cellshas decreased [8-10]. The complete control and eradication of infection isachieved by humoral responses, in which circulating viral particles areneutralized by protective anti-HBs antibodies.
免疫系统对病毒的感染在某些阶段是精心安排的（orchestrated），他们相互一起作用来清除病原体（pathogen）并且让宿主拥有记忆细胞可以对抗以后接下来的感染。在急性乙肝感染的初期，自然杀伤细胞（NK）是宿主对抗的最前线，这而细胞的激活可以通过释放干扰素（interferon (IFN)-γ）帮组降低病毒的含量。但是，在血病毒的最高峰的时候，这些细胞的激活立即被核实并且短暂的伴随一波白细胞间介素-10（IL-10）的出现。在后期的5到6周的后期感染，后天获得性免疫被降低，CD8和CD4 T细胞的数量扩充并且成为控制病毒的主力。HBV特异性的T细胞响应参与到感染肝细胞的清除工作中，这个过程开始于抗病毒细胞因子的释放（干扰素IFN-γ和肿瘤坏死因子(TNF)-α），一旦受感染细胞内的MHC-class I 多肽复合物开始降低，系统随后通过生产细胞毒素（cytotoxic）分子来清除感染的细胞【8-10】。彻底的控制和清除感染是通过体液（humoral）响应来实现的，通过将流动的病毒颗粒被保护性的抗HBs抗体中和。
[attach]220458[/attach]
图一：慢性乙肝患者T细胞响应的损坏T细胞在肝脏内部沿着血窦（sinusoidal）网络流动，在这个网络里他们同肝细胞和呈现抗原的细胞（Kupper细胞和
内皮瘤）接触。在慢性感染乙肝期，CD8T细胞在肝脏内对抗病毒颗粒，病毒抗原和感染的肝原性细胞。他们和感染的肝原性细胞和Kupffer细胞通过T细胞的受体（TCR）-MHC类型I多肽复合物相互作用。在慢性感染期，肝细胞大量表达PD-L1，通过PD-1路径提供给CD8+T细胞一个阻止信号。这个消极的信号导致T细胞功能的损伤：降低扩增，细胞毒素功能和产生抗病毒的细胞因子（IFN-γ TNF-α）。T细胞的耗竭也可以由大量的调控T细胞（Tregs）出现和高水平的IL-10分泌导致。
The functional impairment of immuneresponses is a key feature of chronic HBV infection (Fig.1) [11]. When T cellsencounter HBV antigens presented by intrahepatic antigen-presenting cells, suchas liver-resident dendritic cells (DCs), Kupffer cells or liver sinusoidalendothelial cells, the costimulation signals received by T cells are too weak, drivingthe immune response toward tolerance rather than functional activation. TheIFN-α and IL-8 cytokines produced by hepatocytes and inflammatory cells inliver promote NK cellmediated hepatocyte death and liver damage [12]. T-celldefects are directly related to the sustained exposure of these cells to viralantigens, such as HBsAg and HBeAg, which are produced in large amounts over aperiod of decades during chronic infection. This chronic exposure to antigensleads to the progressive exhaustion of T cells, which lose their effector functions,such as cytokine production (TNF-α and IL-2), cytotoxicity,and proliferation. Two major inhibitory receptors at the cell surface,programmed death-1 (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA-4), havebeen identified as regulators of CD8 and CD4 T-cell effector function [13, 14].PD-1 is overexpressed on HBV-specific T cells, and its level of expression iscorrelated with viral load [15]. PD-1-overexpressing T cells produce only smallamounts IFN-γ and cannot differentiate into memory cells [16]. Ultimately, theHBV-specific T-cell population may be entirely deleted following prolongedexposure to high doses of HBV antigens. For example, CD8 T cells specific fordominant epitopes have been shown to be undetectable in the liver andperipheral blood of patients with HBV viral loads exceeding 107 copies/ml [17].It has also recently been shown that pro-apoptotic genes are more strongly expressedin HBV-specific CD8 T cells from patients with chronic infection than in those frompatients who resolve infection. The molecule most strongly upregulated in thiscontext is the Bcl2-interacting mediator of death (Bim) protein, a member ofthe Bcl2 family that plays a crucial role in the initiation of lymphocyteapoptosis [18].
慢性HBV感染的核心特征是免疫响应系统受到破坏（图一所示）【11】。当T细胞碰到由肝内抗原呈现(antigen-presenting)的HBV抗原，例如基于肝脏（liver-resident）的树突细胞（DCs），Kupffer细胞或者是肝脏窦状隙内皮（sinusoidal endothelial）的细胞，T细胞接收到的共同刺激的信号非常弱，导致免疫反应趋于免疫耐受（tolerance）而不是功能性的激活。由肝内细胞，炎症细胞产生的IFN-γ和IL-8细胞因子促使NK细胞调节肝细胞死亡和肝损伤【12】。由于长期数十年的慢性感染导致的细胞内大量的病毒抗原的累积，比如说HbsAg和HbeAg，T细胞由于持续的对抗这些抗原导致其数量明显的不足。慢性的长期暴露在抗原下面导致进展性的耗竭T细胞，使它们失去了他们的效应（effector）功能,包括细胞因子的生产（TNF-α和IL-2），细胞毒性，和扩增。两种主要的细胞表面的受体：程序死亡1（PD-1）和细胞毒素T淋巴细胞抗原4（CTLA-4），被认为是用来调节CD8和CD4T细胞的效应功能。 PD-1在HBV特异性T细胞中过度表达，它的表达和病毒的载量有关【15】。PD-1过度表达的T细胞仅仅生产少量的IFN-γ并且不能分化成其他的记忆细胞【16】。最终，由于长期的暴露在高剂量的HBV抗原下面，HBV特异性的T细胞的数量可能被彻底的清除。比如说，在病毒的含量超过10^7方的病人肝部和外周血内，针对主要抗原表位（epitopes）的CD8 T细胞几乎已经不可以被检测到【17】。最近显示，慢性感染患者的 HBV特异性的CD8 T细胞比治愈的患者拥有更强的表达pro-细胞凋亡（pro-apoptotic）的基因。在整个系统中，最强烈上调（upregulated）的分子是Bcl2-相互作用调节的死亡（Bim）蛋白，一个在初始淋巴细胞的凋亡上面起着重要作用的归属于Bcl2家族的蛋白【18】。
Other major actors potentially involved in the global collapse ofimmune responses during chronic hepatitis B infection include regulatory Tcells (Tregs) and DCs. Patients with a high HBV load have a high proportion ofTregs in the liver, but not in blood [19, 20]. However, the role of Tregs inthe modulation of CD8 T-cell effector functions, and the contribution of thesecells to T-cell dysfunction in vivo remain a matter of debate [21, 22]. Thehigh frequency of Tregs in the liver may also be a direct consequence of liver inflammation,because these cells are induced to protect organs against strong, potentially damagingimmune responses. Tregs have therefore never been considered as possibletargets for the treatment of chronic hepatitis B. As professional antigen-presentingcells, DCs play an important role in the induction of functional T cells.Patients with chronic HBV infection display not only a decrease in the numberof circulating DCs, but also functional defects in these cells that maycontribute to the dysfunction of HBV-specific T cells [23]. Functional impairmentof both myeloid (mDCs) and plasmacytoid DCs (pDCs) from patients withHBVrelated chronic hepatitis has been described [24]. However, it has beenshown that DCs from such patients cannot support HBV replication, despite beingable to take up viral antigens and viral DNA, ruling out a direct role of viralreplication [25]. Recent data have shown that B7-H1 (PD-L1), the known ligandof PD-1, is upregulated on mDCs from patients with chronic liver inflammation,including patients with chronic hepatitis B [26]. It remains to be determinedwhether the T-cell defects in these patients are due to inhibitory signals transmittedto the T cells by DCs over-expressing PD-L1 or to liver inflammation.
另一个主要的潜在参与慢性乙肝病毒感染导致的全局性的免疫反应坍塌包括调节性T细胞（Tregs： regulatory T cells）和DCs。具有高HBV病毒载量的病人在肝部有大量的Tregs，但是血液中却没有【19，20】。然而，Tregs在调节CD8 T细胞的效应功能的作用，还有其在导致T细胞机能障碍上的贡献都存在一定程度的争论【21,22】。Tregs在肝脏中的高频率可能也直接导致肝脏的炎症，因为这些细胞的产生是为了保护器官，由于对抗强的，潜在的破坏性的免疫反应。Tregs因此也从来没有被认为是一个治愈乙肝的可能的靶位。作为专业的抗原呈现（antigen-presenting）细胞，DCs起到了一个非常重要的诱发（induction）功能性T细胞的角色。慢性乙肝患者体现了不仅是流动的DCs细胞的降低，而且这些细胞的功能性上的缺陷可能导致HBV特异的T细胞的功能障碍【23】。在HBV慢性感染者中，可以发现脊髓的树突细胞（mDCs）和类浆树突细胞（pDCs）功能性的的损伤【24】。然而从这些病人中的DC细胞不可以支持HBV的复制，不管这些DC细胞可以吸收（take up）病毒的抗原和病毒DNA,，这样排除了（ruling out）一个直接的病毒复制角色。近期的数据显示B7-H1（PD-L1），一个已知的PD-1的配体，在慢性炎症肝炎包括慢性乙肝的mDCs细胞中是上调的【26】。目前仍然不清楚的是到底在这些患者中T细胞的瑕疵是由于DCs细胞过多表达的PD-L1渗透到T细胞，还是由于肝脏的炎症引起。
The functional changes to the immune system, which have onlyrecently been discovered, may account for the poor results obtained duringinitial attempts to develop therapeutic vaccines [27, 28]. Improvements in ourunderstanding of the role of the immune response in the control of viralinfection and liver damage, will facilitate the development of new strategiesbased on immunotherapy that, when combined with current antiviral treatments,could accelerate viral clearance, making life-long treatment unnecessary(Table1).
最近才刚刚发现，免疫系统功能性的改变，可能可以解释为什么治疗性疫苗的初始结果不是那么的好【27,28】。
对免疫反应在控制病毒感染和肝脏损伤的进一步认识，可以帮助开发新的基于免疫治疗的策略，当这种策略和现有的抗病毒治疗相结合，可以加快病毒的清除，从而实现避免终身治疗（表1）。
表一：慢性乙肝感染的特点和当前或将来可以用于改善免疫调节控制病毒感染的策略
| 慢性乙肝感染的特点 | 治疗目标 | 工具 | 预期结构 |
高病毒载量 | 降低病毒和抗原载量 | 核苷类 | 短暂的恢复T细胞功能 |
HBV特异性T细胞的高水平的PD-1表达 | 降低阻止信号 | 抗PDL-1抗体 | 恢复T细胞功能 |
HBV特异性T细胞的耗竭 | 活体从新激活/激活HBV特异性T细胞 | -强大的疫苗 -新的佐剂 -DNA疫苗 -病毒疫苗 -抗原抗体复合物（Ag/Ab IC） -细胞因子 | -从新激活Th1响应 -从新激活CD4和CD8T细胞 -改善抗原的出现 -T细胞扩增 |
体外激活T细胞 | 修改的DCs-PBMCs激活 | -改善抗原呈现 -激活杀伤细胞 | |
HBV特异性T细胞清除 | 改造新的HBV特异性T细胞 | TCR重新导向T细胞 | -无耗竭，功能性HBV特异性T细胞 -识别HBV感染的细胞 |
抑制的环境 | 靶向非HBV特异性的T细胞到肝脏 | 肝脏基因治疗 | 清除HBV感染的肝原性细胞 |
The first therapeutic vaccine trials were based on prophylacticrecombinant vaccines containing HBV envelope proteins carrying HBsAg (Table 2).This was a logical initial choice, because the final goals of any anti-HBVtreatment are the clearance of HBsAg and the induction of protective anti-HBsantibodies. Pilot clinical studies demonstrated that standard vaccinationinduced a significant increase in HBeAg seroconversion [27]. Although the percentageof serum samples in which HBV DNA levels decreased or became undetectable washigher in some vaccinated groups than in control groups, the effects were notsustained. HBV vaccine-specific T-cell responses, mostly involving CD4 T cells,were also induced. However, few patients displayed HBsAg clearance and anti-HBsseroconversion, highlighting the need for vaccines with a greater therapeuticpotential.
第一个治疗性疫苗的测试是基于预防性的组合含有HbsAg 的HBV封装包膜蛋白(envelope protein)（表2）。这是一个逻辑上的初始选择，因为所有的抗HBV治疗的最终目的是为了清除HbsAg和产生保护性的抗Hbs抗体。前期试探性的临床研究显示普通的疫苗可以明显提高HbeAg的血清转换【27】。尽管在疫苗组大部分病人的血清中HBV-DNA下降或者是到不可被检测，这个数据比空白对照组要高，但是这个效果却没有持续性。HBV疫苗特异的T细胞响应，主要涉及到CD4 T细胞也被诱导产生。但是，少数病人显示HbsAg清除并且出去抗Hbs血清转换，提示对疫苗的更好的治疗潜力的需求。
Several ways of increasing vaccine efficacy have been proposed,including changes in vaccine composition and in the route used for vaccinedelivery. A double-blind placebocontrolled Phase II B clinical trial has beencarried out on 242 patients with chronic hepatitis B infection. These patientsreceived injections of antigen-antibody-complexes (HBsAg-anti-HBs immunecomplexes (IC)) with alum as the adjuvant, with the aim of targeting DCs. DCs incubatedwith IC secrete large amounts of IL-12. They upregulate functional markers invitro and are thought to prime CD8 T cells in vivo. A significant virologicaleffect was observed 24 weeks after the last of six IC injections. The HBeAgseroconversion rate was 21.8% in the group immunized with 60 μg IC, and only 9%in the control group immunized with alum
alone [29]. However, the immuneresponses accounting for this therapeutic effect were not
analyzed in this study.
几种提高疫苗效率的途径被提出，包括改变疫苗的组分和疫苗的运输方式。一个针对242个慢性乙肝病人的双盲PhaseII B实验已经展开。这些病人接受抗原-抗体复合物（HbsAg-抗Hbs免疫复合物（IC））并带有铝剂（alum）作为辅剂进行治疗，目标是为了靶向DCs。DCs和IC一起孵化分泌大量的IL-12.在体外实验中，他们上调功能性的标记物，并被认为在活体中启动CD8 T细胞。在最后6次IC注射完之后，一个显著的病毒学效应在24周后被发现。在注射 60 ug IC的小组显示HbeAg血清转换率在21.8%，
然而在空白对照组仅在9%【29】。另外，在这项研究中，导致这种治疗效果的免疫反应没有深入分析。
Most of the therapeutic HBV vaccines designed to date have usedenvelope proteins as the target antigen, but core antigen (HBcAg) and thepolymerase are also specific targets of the immune response duringself-limiting hepatitis B. A novel vaccination approach, based on the use of acombination of recombinant HBsAg and HBcAg and known as “NASVAC”, is currentlybeing developed for both the prevention and cure of hepatitis B. Both theseantigens form virus-like particles. When they are mixed together, they formhighly immunogenic superstructures, with the two antigens working together in thedevelopment of T- and B-cell responses [30]. This novel vaccine formulation hasbeen administered intranasally to individuals taking part in a phase I trial[31]. The vaccine was found to be safe and immunogenic, eliciting anti-HBc andanti-HBs seroconversion. Preliminary results of the ongoing Phase I/II trial inpatients with chronic hepatitis B infection in Bangladesh were presented at“The 20th conference of the Asian Pacific association for the study of theliver”[32].
目前绝大多数设计的HBV治疗性疫苗都是用封装包膜蛋白作为目标抗原，但是在自封闭乙肝中，HbcAg和聚合酶同样也是免疫反应的特异靶位。一种新型的疫苗方案，基于使用HbsAg和HBcAg的组合
命名为‘NASVAC’的疫苗，已经正在被开发用于预防和治疗乙肝。所有这些抗原都形成病毒性颗粒。当他们被混合在一起，他们形成高度免疫原性的（immunogenic）超级结构，两种抗原一起工作用于发展T细胞和B细胞的响应【30】。这种新型的疫苗方案已经被批准用于Phase I 临床测试【31】。这个疫苗被发现是安全的和具有免疫原性的，可以激发（eliciting）抗Hbc和抗Hbs的血清转换。在第20届亚太地区肝病研究组织会议上，针对孟加拉慢性乙肝感染患者已经有正在进行的PhaseI/II期的初步数据【32】。
A decrease in HBV load seems to precede the detection ofHBV-specific T-cell responses, both in patients resolving natural infectionsand in those displaying flare-ups of hepatitis associated with HBeAgseroconversion during chronic infection. Reducing HBV load by antiviralchemotherapy may therefore increase the responsiveness of HBV-specific T cells,which are hyporesponsive in cases of persistent HBV or viral antigenstimulation. Indeed, HBV-specific T cells are detectable during the first fewmonths of lamivudine treatment [33]. However, this restoration of T-cellactivity is partial and transient and does not
lead to an increase in HBeAg seroconversion[34]. Classical preventive recombinantvaccines do not themselves have a strong antiviral potential, but pilot studieshave shown that they are most effective in patients with a low HBV load at thestart of treatment [27]. Rational therapeutic approaches based on thesefindings have been developed. They combine the vaccine-induced exogenousstimulation of protective T- and B-cell responses with the concomitantsuppression of viral replication by antiviral drug treatment. Proof-of-concepthas been demonstrated in animal models, notably duck and woodchuck, in which acombination of antiviral drugs and vaccination had sustained therapeuticeffects [35-37] (Table 2).
在自然自愈病毒感染的和慢性感染带有病毒骤发（flare-ups）并伴随HbeAg血清转换的病人中, 都可以发现病毒载量的下降看起来可以导致HBV特异性的T细胞响应被检测到。通过抗病毒化学疗法降低病毒的载量可能导致增加HBV特异性的T细胞反应，而这些细胞在持续HBV或者病毒抗原刺激下是低反应的（hyporesponsive）。
事实上，在拉米夫定的初期治疗的几个月内，HBV特异性的T细胞是可以被检测到的【33】。
然而，恢复T细胞的过程是不完全的而且持续很短暂，并不能导致增加HbeAg血清转换【34】。传统的预防性组合疫苗自己本身并没有很强的抗病毒能力，但是他们在对于那些拥有较低病毒载量的患者更加有效【27】。基于这些发现，合理的治疗方案已经在开发。他们组合由疫苗导致的外生(exogenous)刺激保护性T细胞和B细胞的响应同时伴随（concomitant）抗病毒治疗的药物对病毒复制进行压制。这个概念已经在动物模型中得到演示，比如说鸭和土拨鼠模型，组合使用抗病毒药物和疫苗可以支撑治疗的效果【35-37】（表2）.
Lamivudine is the nucleoside analog most widely combined withmultiple administrations of vaccine. In most published clinical trials, effortsto prevent the emergence of escape mutants during the prolonged use oflamivudine have been based on the use of this analog for a short period only(six to 12 months). The combination of vaccine and lamivudine is welltolerated, with no serious adverse effects. Responses to combination therapyhave generally been evaluated on the basis of virological and serologicalparameters, such as the decrease in HBV DNA levels, the loss of HBeAg and HBsAgand seroconversion. Many of the patients enrolled in clinical trials have neverbeen treated before and have high serum ALT levels. Serum ALT levels are animportant predictor of the response to lamivudine treatment [38]. In two recentstudies, following six months of combination therapy, regardless of thecomposition of the vaccine, HBeAg/anti-HBe seroconversion was described inabout 25% of treated patients [39, 40]. These results should be compared withthe current rate of HBeAg seroconversion in lamivudine-treated patients ofabout 15%. In one study, patients were followed for more than 6 years followingcessation of treatment. Twenty-four % of them were classified as sustainedresponders with HBV DNA below detection limit, persistence of anti-HBe and norelapse in hepatitis [40]. However, this study had two major drawbacks. First,it has no control group and second, the technique used for HBV-DNAquantification was less sensitive than that used in other trials. This raisedthe question of whether the virus is really cleared or just decreased to belowdetection limit.
拉米夫定是核苷类抗病毒药物被广泛的和各种疫苗使用。在大部分发表的临床试验中，为了阻止出现耐药的变异体，拉米夫定仅被使用了短暂的时间（6到12个月）。组合使用拉米夫定和疫苗耐受性好，而且没有严重的副作用。组合治疗的响应主要通过病毒学和血清学上面的参数来评价，比如说HBV-DNA的下降，HbeAg和HbsAg的消失和血清转换。很多参加的临床试验的病人以前都没有接受过治疗并且有高的
血清转氨酶水平。血清Alt水平是一个非常重要的拉米夫定响应的预言者【38】。在近期的两项研究中，经过6个月的组合治疗，不管疫苗的成分，HbeAg/抗Hbe血清转换率大概在25%【39,40】。而单独拉米夫定治疗的HbeAg血清转换在15%。在一项研究中，患者在超过停止6年之后随访。24% 被归类于持续性的响应者，包括HBV-DNA低于检测限，持续的抗Hbe出现而且肝炎没有反复【40】。然而，这个研究有两个不足之处。第一，没有空白对比试验，第二，HBV-DNA的量化的灵敏度没有其他试验使用的高。这提出了问题：是否病毒真的被清除还是仅仅是降到检测限一下？
In a randomized controlled study of 180 patients assigned to threegroups, Hoa et al. evaluated the relative contributions of a preventive vaccineand antiviral drugs to the treatment of chronic HBV patients during activedisease. Patients received vaccine monotherapy, lamivudine monotherapy or acombination treatment. The combination treatment was found to givesignificantly higher and earlier rates of viral suppression than lamivudine orvaccine monotherapy. HBeAg seroconversion rates did not differ significantly betweenthe three groups of patients. However, a continual increase in the rate of seroconversionwas observed in patients from the vaccinated groups, suggesting that the vaccineenhances the antiviral immune response [41]. Results of these trials have to becompared with those of a randomized controlled study in which lamivudine wasgiven with a hepatitis B vaccine mixed with a strong Th-1 adjuvant [42]. Inthis study, despite 12 injections of vaccine over a one year period, neitherimprovement in HBe-seroconversion rate nor decrease in HBV-DNA levels wereobserved in the vaccine group compared to treatment with lamivudine alone.
在一份随机对照的研究中，180位患者被分为3个组，Hoa et al. 用于评价抗病毒药物和预防性疫苗对慢性活动期HBV患者治疗的贡献。患者被分别使用单一的疫苗，单一的拉米夫定，或者是两者的组合治疗。组合治疗被发现可以提供明显的更高的和更早的病毒压制比率
相对于拉米夫定或者是疫苗单独治疗。三组中，HbeAg血清转换率并没有明显的不一样。然而，在疫苗组可以发现持续的血清转换率的增加，提示疫苗增加了抗病毒的免疫反应【41】。这些实验的结果可以和另一项随机控制的研究作比较，这项研究中拉米夫定和一种乙肝疫苗一起使用，但同时增加了强的Th-1作为佐药（adjuvant）【42】。在这项研究中，不管在一年的时间内注射了12针疫苗，相对于拉米夫定组，疫苗组没有发生改进的Hbe血清转换或者是降低HBV-DNA（ps，针对于拉米夫定组而言，两者没有区别？）。
In combination treatment, the time between the administration of theantiviral drug and that of the therapeutic vaccine also appears to be crucialfor the immune responses induced. T-cell restoration upon lamivudine treatmentaccompanies the decrease in viral replication, but this effect disappears aftersix months, even in cases of continuous antiviral treatment [34]. The CD8T-cell response, to HBcAg in particular, is also closely correlated with viralload [17]. It would therefore seem logical to begin vaccination when serum HBV DNAlevels have just become undetectable. The results obtained for patients withchronic hepatitis B undergoing an intradermal vaccination program with anHBsAg-based preventive
vaccine beginning three months after theinitiation of antiviral treatment provide support for this strategy. Indeed,the rate of seroconversion from HBeAg to anti-HBe was 56% for the combinationregimen, whereas it was only 16% for lamivudine monotherapy [43]. It should benoted that this high rate of seroconversion was significantly associated with aninitial low viral load. Interestingly, none of the patients receiving thecombination therapy displayed HBV DNA or hepatitis breakthrough during thethree-month post-treatment follow-up period of the study, whereas such problemswere observed in the monotherapy group and have been reported in other studies.
在一个组合治疗中，给予抗病毒药物和治疗性疫苗治疗的时机对免疫响应非常重要。在拉米夫定治疗中， T细胞的恢复伴随着病毒复制率的下降，但是这个效果仅仅出现在6个月之后，即使在持续的抗病毒治疗的例子里面【34】。CD8 T细胞的响应，尤其是对HBcAg的响应，也直接和病毒的载量有关【17】。因此可以看到，只有当HBV-DNA下降到不可检测的程度时候注射疫苗在逻辑上才有效。在慢性乙肝患者抗病毒治疗后的3个月，进行皮下疫苗注射基于HbsAg预防性疫苗项目的实验结果也支持这种策略。事实上，组合治疗法给出了HbeAg血清转换在56%以上，然而单独拉米夫定治疗仅在16%【43】。必须指出的是高比率的血清转换非常明显的和最开始治疗前低病毒载量有关。有意思的是，在治疗后的随后的3个月的随访研究中，没有病人显示HBV-DNA和肝炎暴发，而这些问题在单独治疗的小组却被发现。
HBV DNA loss or decrease were generally used as end point to assessviral control in the various studies. However, it is difficult to reconcile theresults as none of these studies used the same criteria to define virologicalresponders. In addition, the patients enrolled in the clinical trials may havea completely different disease history depending on the mode of virus transmission,the age at which contamination occurred and duration of chronic infection.
HBV DNA减少或者是降低通常被用于各项研究中的病毒控制的终极目标。但是，对于病毒学上的响应很难定义一个相同的标准来比较不同实验得到的结果。此外，参与临床的病人可能拥有不同模式的疾病历史决定于乙肝感染的途径，慢性感染持续的时间，第一次被感染的年龄。
Only a few reports have documented humoral or cellular immuneresponses. The humoral response has been assessed through the detection ofanti-HBs antibodies, whereas proliferation and cytokine production have beenused as indicators of cellular responses. Vaccination clearly stimulates thehumoral response, because 50 to 85% of vaccinated patients with chronic HBVinfection develop antibodies against HBsAg. However, HBsAg clearance has onlyrarely been observed [41, 42]. Furthermore, although the HBsAg-specific lymphoproliferativeresponses in patients with chronic hepatitis B were found to be similar to thosein healthy vaccine-recipients, the T cells of these patients secreted lessIFN-γ. This suggests that the anticipated Th1 stimulation in response tovaccination may not be achieved in this setting [42]. In addition, noassociation was found between HBsAg-specific T-cell proliferation and anyclinical or virological improvement in the few patients for which immuneresponse was analyzed.
仅仅有少数报告记载了体液和细胞的免疫反应。体液反应可以认为从检测到抗Hbs抗体开始，扩增和细胞因子的生产被认为是细胞响应的指示剂。疫苗明显的刺激了体液响应，因为50%到85%接受过疫苗的慢性乙肝患者产生了抵抗抗体。然而，HbsAg的清除仅仅是少数观察【41,42】。另外，尽管慢性乙肝患者可以发现针对HbsAg特异的淋巴组织增生反应，这和健康疫苗接种者一样，但是这些患者的T细胞分泌更少的IFN-γ。这提示预期的Th1在免疫反应的激活可能在这种设定下面不能达到【42】。另外，少数病人身上的免疫反应被分析，但是HbsAg特异性T细胞的扩增和任意临床或病毒学的改善之间没有联系。
The use of strong Th1-inducing adjuvants or cytokines might increasethe efficacy of therapeutic vaccination. Several trials have evaluated the useof a therapeutic vaccine combined with IL-2 to treat human immunodeficiencyvirus of HBV infection [44, 45]. The withdrawal of antiviral treatment at theend of vaccination had almost no effect on the risk of viremia relapse.However, one potential drawback of IL-2 treatment is that it induces Tregs, whichconstitutively express IL-2Rα. Other cytokines, such as IL-7 and IL-15, areessential for the homeostatic proliferation of T cells in vivo, the survival ofeffector cells and the
maintenance of memory T cells during viralinfection. These cytokines may increase the pool of naïve T cells available forvaccine-mediated stimulation and the survival of activated T cells. Studies onanimals have shown that IL-7 improves anti-tumor responses and survival after avaccine-induced immune response [46]. A clinical trial combining antiviraltreatment with a classical preventive vaccine and multiple IL-7 injections iscurrently underway in HBenegative chronic hepatitis B patients with nodetectable HBV DNA during at least three months of anti-viral treatment (www.ClinicalTrials.gov,Identifier: NCT01027065).
使用强的Th1导致的佐剂或者是细胞因子可能可以增加治疗性疫苗的效率。几组实验已经用于评价治疗型疫苗和IL-2组合效果治疗人免疫缺陷病毒感染的HBV感染【44,45】. 疫苗治疗后期，取消抗病毒药物的治疗几乎不会产生病毒学反弹。然而，一个潜在的坏处是TL-2治疗导致Tregs的产生，这个可以持续的表达IL-2R。其他的细胞因子，在活体实验中比如说IL-7和IL-15对自我平衡（homeostatic）的扩增T细胞是必须的，
同时对效应细胞的存活和维持病毒感染期间记忆T细胞也是必须的。这些细胞因子可能增加幼小T细胞的含量，而这些幼小T细胞可以用于疫苗调节激发和激活的T细胞的存活。动物研究显示，在疫苗导致的免疫响应以后，IL-7改善了抗肿瘤响应和存活【46】。一项临床研究结合抗病毒治疗和传统的预防性疫苗，并带有数次IL-7注射正在Hbe阴性并且HBV-DNA在接受至少3个月的抗病毒治疗达到低于检测限的慢性乙肝患者身上展开(www.ClinicalTrials.gov,Identifier: NCT01027065).。
Experimentsin mice with persistent lymphocytic choriomeningitis virus infection have shownthat anti-PD-L1 antibodies and therapeutic vaccination synergistically improve viralcontrol [47], by blocking the PD-1/PD-L1 inhibitory signals on exhausted T cells.In vitro studies have shown that it may be possible to restore intrahepaticHBV-specific T-cell responses at least partly in patients with chronic HBV infection,by blocking the PD-1 pathway [15]. Targeting of the PD-1 pathway, incombination with vaccination and antiviral treatments, therefore constitutesanother possible approach to restoring T-cell function and generating long-termmemory T cells [48].
在长期受淋巴细胞（lymphocytic）脉络丛脑膜炎（choriomeningitis）病毒感染的小鼠实验中显示，通过阻断PD-1/PD-L1耗竭了的T细胞的抑制信号，anti-PD-L1抗体和治疗性疫苗协同的（synergistically）改善对病毒的控制【47】，。一些体外实验显示，通过阻止PD-1的路径，有可能可以恢复至少在部分慢性乙肝感染患者肝内HBV特异性的T细胞响应【15】。靶向PD-1的路径，同时结合疫苗和抗病毒治疗，这样这组成了另外一种可能的途径去恢复T细胞的功能和产生长期记忆性的T细胞【48】。
DNA-basedvaccines are increasingly being tested, for both preventive and therapeutic vaccinationpurposes, and encouraging results have been obtained in mice and chimpanzees [49,50]. The functional profile of the immune responses generated by immunizationwith plasmid DNA is similar to that observed in patients with resolved HBVinfection and selflimited disease [51]. This type of vaccination has theadvantage of inducing both humoral and cellular immune responses, includingcytotoxic and Th1 responses [52].
基于DNA的疫苗的测试正在增加，包括以预防性和治疗性为目的的疫苗，鼓舞人心的结果已经从老鼠和猩猩得到【49,50】。由质粒DNA导致的免疫反应的功能性内容和那些自发自愈乙肝的患者和（self limited disease）自治愈疾病中的发现是一样的【51】。这种类型的疫苗的优势是既可以激发体液免疫也可以激发细胞免疫，包括细胞毒素和Th1响应【52】。
临床前期的在动物模型中的研究
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Recombinant HBV envelope proteins (HBsAg) withadjuvants in HBV/HBsAg
transgenicmice [74-76]
DNA vaccines encoding HBV envelope proteins or polymerase in HBsAg/HBV
transgenicmice [49, 77-79]
Proteins or DNA vaccinescoding for envelope orcore proteins of woodchuck
hepatitis virus or +/- antivirals in chronically infected woodchuck [36, 37, 80]
DNA vaccines encoding DHBV large envelope protein +/- antivirals inchronically
infectedducks [35, 81]
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Past vaccine trials/以往的临床测试
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Preventive hepatitis Bvaccines# [82, 83, 27]
Preventive hepatitis Bvaccines + antiviral treatment(lamivudine) [39-41,43] + Th-
1 adjuvant [42]or IL-2 [45]
DNA vaccines* +/- antiviral treatment [53, 55]
HBsAg/anti-HBs immunecomplexes¶ [29]
Cell-based therapies:CIK° cells [68]; DC+ HBV antigens§ [62, 64]
Ongoing clinical trials/进行中的临床测试
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Recombinant hepatitis Bproteins (HBsAg+HBcAg), nasal immunization [30, 32];
Preventive hepatitis B vaccine +/- Clevudine ( NCT00501124)
DNA vaccines*+ antiviral treatment (NCT00536627; NCT00513968; NCT01189656)
Preventive hepatitis Bvaccine (preS2/HBsAg vaccine) + IL-7 + Entecavir or
Tenofovir (NCT01027065)
HBsAg/anti-HBs immunecomplexes¶ [84]
Future approaches/将来的方案
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• Anti-PD-L1 antibodies [15]
• DNA+ viral vectors (prime-boost strategy) [85, 86]
• TCR-transduced T cells with HBV specificity [70, 71]
• HBV-based gene therapy [72]
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# Recombinant HBsAg, preS2/HBsAg orpreS1/preS2/HBsAg with Alum as adjuvant
* DNAs encoding HBV proteins (DNAencoding small and middle envelope proteins
or mixed DNAs encoding envelope,capsid and polymerase proteins and human IL-
12)
¶ HBsAgcomplexed to human anti-HBs immunoglobulins with Alum as adjuvant
°Cytokine-induced killer cells
§ Dendriticcells loaded with HBsAg or HBV-derived peptides
NCT : ClinicalTrials.gov Identifier(http://clinicaltrials.gov)
The first Phase I trial of therapeutic DNA immunization for chronichepatitis B was carried out with a DNA vaccine encoding HBV envelope proteins[53] (Table 2). At the time of inclusion, the treated HBV patients presentedviral breakthrough on lamivudine and active HBV replication. Despite thisunfavorable environment, a decrease in viral load and HBeAg seroconversion wereobserved in six and two of 10 patients, respectively. Seroconversion occurredin the two patients with the lowest viral load at the beginning of the trial, highlightingthe importance of HBV DNA titers for successful immunotherapy. DNA vaccinationled to the induction of IFN-γ-secreting T cells specific to severalenvelopederived epitopes and cytotoxic CD8+ T cells, demonstrating the abilityof this approach to induce or to increase the number of HBV-specific T cells.This HBV-specific IFN-γ T-cell response was also correlated with an increase inthe percentage of a specific NK-cell subset known to produce cytokines inabundance, the CD56bright NK-cell population [54]. Nevertheless, despite thestimulation of innate and adaptive immune responses, no severe adverse eventswere reported, with the exception of a transient increase in ALT levels in two patients,probably due to the restoration of T-cell responses. However, T-cell reactivitygradually declined at the end of treatment, reflecting the profound defect inimmune responses observed during chronic HBV infection.
第一个临床I期的治疗性针对慢性乙肝的DNA疫苗已经展开，DNA疫苗是带有表达HBV封装包膜蛋白的基因【53】（表二）在时间上的包容（At the time ofinclusion），拉米夫定治疗的HBV患者出现了病毒突破和病毒重新活跃复制。除了这个不友好的环境外，10人中六人病毒的载量下降，10人中的两人发生了HbeAg血清转换。发生血清转换的两个病人开始试验前含有最少的病毒载量，提示HBV DNA的定量是免疫治疗成功的关键。DNA疫苗导致IFN-γ分泌针对几个由病毒外壳延伸的抗原表位特异性的T细胞和细胞毒素CD8+ T细胞，显示这种方法有能力导致或者增加HBV特异性的T细胞。这些HBV特异的IFN—γ分泌的T细胞的响应也和NK-细胞的子集（一种叫做CD56 brightNK细胞的群体，他们可以产生丰富的细胞因子）的增加有关。尽管如此，不考虑先天和后天获得性免疫反应的激活，没有发生严重的副作用，其中有两个患者的转氨酶有预期的短暂的升高，可能是因为T细胞响应的恢复导致的。然而，在实验的最后T细胞的活性缓慢的下降，反应了慢性HBV感染中导致的免疫反应的深度的破坏。
The first Phase I trial of therapeutic DNA immunization for chronichepatitis B was carried out with a DNA vaccine encoding HBV envelope proteins[53] (Table 2). At the time of inclusion, the treated HBV patients presentedviral breakthrough on lamivudine and active HBV replication. Despite thisunfavorable environment, a decrease in viral load and HBeAg seroconversion wereobserved in six and two of 10 patients, respectively. Seroconversion occurredin the two patients with the lowest viral load at the beginning of the trial, highlightingthe importance of HBV DNA titers for successful immunotherapy. DNA vaccinationled to the induction of IFN-γ-secreting T cells specific to severalenvelopederived epitopes and cytotoxic CD8+ T cells, demonstrating the abilityof this approach to induce or to increase the number of HBV-specific T cells.This HBV-specific IFN-γ T-cell response was also correlated with an increase inthe percentage of a specific NK-cell subset known to produce cytokines inabundance, the CD56bright NK-cell population [54]. Nevertheless, despite thestimulation of innate and adaptive immune responses, no severe adverse eventswere reported, with the exception of a transient increase in ALT levels in two patients,probably due to the restoration of T-cell responses. However, T-cell reactivitygradually declined at the end of treatment, reflecting the profound defect inimmune responses observed during chronic HBV infection.第一个临床I期的治疗性针对慢性乙肝的DNA疫苗已经展开，DNA疫苗是带有表达HBV封装包膜蛋白的基因【53】（表二）在时间上的包容（At the time ofinclusion），拉米夫定治疗的HBV患者出现了病毒突破和病毒重新活跃复制。除了这个不友好的环境外，10人中六人病毒的载量下降，10人中的两人发生了HbeAg血清转换。发生血清转换的两个病人开始试验前含有最少的病毒载量，提示HBV DNA的定量是免疫治疗成功的关键。DNA疫苗导致IFN-γ分泌针对几个由病毒外壳延伸的抗原表位特异性的T细胞和细胞毒素CD8+ T细胞，显示这种方法有能力导致或者增加HBV特异性的T细胞。这些HBV特异的IFN—γ分泌的T细胞的响应也和NK-细胞的子集（一种叫做CD56 brightNK细胞的群体，他们可以产生丰富的细胞因子）的增加有关。尽管如此，不考虑先天和后天获得性免疫反应的激活，没有发生严重的副作用，其中有两个患者的转氨酶有预期的短暂的升高，可能是因为T细胞响应的恢复导致的。然而，在实验的最后T细胞的活性缓慢的下降，反应了慢性HBV感染中导致的免疫反应的深度的破坏。[attach]220459[/attach]
图二通常的疫苗和抗病毒治疗的组合方案示意图在组合治疗的测试中，慢性乙肝患者被分为两组（A和B）。两组患者都接受抗病毒治疗。在抗病毒治疗的初级，当病毒的含量低于检测限的时候，A组将多次注射疫苗。HBVDNA病毒载量，HbeAg和HbsAg血清转换率被评价。在抗病毒治疗期间，病毒和抗原的载量下降并且功能障碍的HBV特异性T细胞预期应该恢复。在临床实验的末期（注射完最后疫苗之后），两组同时停止治疗，转氨酶水平被连续监测用于保证快速的检测肝脏损伤。B组中预期会有病毒反弹，但是A组应该可以得到血清病毒的控制。在A组患者中，HBV特异性的疫苗激活的T细胞被预期能够施展他们的抗病毒功能包括分泌细胞因子（IFN-γ， IL-2和TNF-α）并且实现扩增。
Similarly, DNA vaccines encoding multiple HBV proteins and amodified human IL-12 molecule were administered once monthly, for 12 months, incombination with lamivudine treatment, to improve the efficacy of DNAvaccination and to broaden immune responses[55]. IL-12 promotes Th1 celldevelopment, cell-mediated cytotoxicity and IFN-γ production and is involved inthe control of viremia in HBeAg-positive chronic hepatitis B carriers [56]. Avirological response was observed in half the treated patients at the end ofcombination therapy and this was significantly higher compared to lamivudinemonotherapy in historical control groups. Memory T cells were detected invirological responders, persisted for 40 weeks after vaccination and wereclosely associated with the suppression of viral rebound after treatment wasstopped. Two of the virological responders still had undetectable viral load 3years after cessation of combined therapy [57]. The presence of modified IL-12as a genetic adjuvant may have contributed as well to the induction oflong-lasting memory T cells. Whether the HBV-specific T cells were present inpatients at the beginning of the trial and were reactivated by the combinedtreatment or were de novo activated by the vaccine is not known as pretreatmentsamples were not analyzed.
相同的，每月给予一次带有多种HBV蛋白和修饰过的人的IL-12分子的DNA疫苗，在12个月内，结合拉米夫定治疗，用于增加DNA疫苗的有效性和扩大免疫反应【55】。IL-12 促使Th1 细胞的发展，细胞调节的细胞毒素和IFN-γ的产生，参与到控制HbeAg阳性乙肝患者的血清病毒中【56】。
在半数接受联合治疗结束时的患者中出现病毒学响应
，这个比率比历史上单独拉米夫定治疗的效果要高很多。在病毒学响应的患者体内可以检测到记忆T细胞，并且记忆T细胞在疫苗之后持续大概40周，并且紧密的和停止治疗后的对病毒反弹的压制相关。在停止治疗后的3年，其中两个患者依然检测不到病毒含量【57】。使用修饰过的IL-12作为基因佐剂可能也对导致长期持续的记忆T细胞有贡献。HBV特异性的T细胞是一开始随着实验出现在患者体内还是由于组合治疗而激活的，或者是由疫苗重新（de novo）激活的还不清楚，因为样本在接受治疗前没有进行分析。
Finally, DNA vaccines may be used to prime immunity before boostingwith viral vectors, such as poxviruses or adenoviruses encoding the sameantigenic proteins. Such combinations result in very large numbers ofmultifunctional T cells, as demonstrated in trials of vaccines for malaria andtuberculosis [58].
最后，DNA疫苗可能可以用于初始免疫，后面可以通过和病毒载体结合提高效果，载体可以包括编译相同的抗原蛋白的水痘病毒（poxviruses）或者是腺病毒。这种组合可以导致大量的多功能的T细胞，这在疟疾和肺结核（tuberculosis）疫苗测试中显示出来【58】。As an alternative to vaccination, DCs loaded ex vivo with HBVantigens could also be used to stimulate T cells (Table 2). The use ofautologous DCs pulsed with tumor-related antigens as immunotherapy for cancerhas been largely documented [59]. Fewstudies have reported the use of DCs for diseases related to viral infections[60], [61]. A pilot study was carried out in five patients with chronic hepatitisB, to evaluate the safety of HBsAg-pulsed autologous DCs [62]. Monocyte-derivedDCs were isolated from peripheral blood from a given patient by a classicalmethod using granulocyte macrophage colony-stimulating factor and IL-4. Theywere then pulsed with purified HBsAg and re-injected into the patient. This approachhad previously been shown to induce anti-HBs antibodies when administered to vaccine-non-responders[63]. Administration was safe and no exacerbation of liver damage was observedin patients with chronic hepatitis B. In another recent trial, autologous DCs pulsedwith HLA-A2-restricted peptides derived from HBcAg (core 18-27 epitope) andfrom the N-terminal part of the middle envelope protein were re-injected on 9occasions into 380 chronic hepatitis B patients in an autologous manner [64].Interestingly, a significant response, as demonstrated by a decrease in HBV DNAlevels to below 103cp/ml and transaminase normalization, was observed in halfthe HBeAg-negative/anti-HBe-positive patients, whereas only 13% of theHBeAg-positive patients displayed complete responses. This treatment was mostsuccessful in HBeAg-negative patients and patients with a viral load <105cp/ml, some of whom had cleared HBsAg. A transient increase in ALT levels was observedin some patients during the first few weeks of treatment, possibly reflectingan effect of the treatment on infected hepatocytes, but ALT levels returned tonormal by the end of treatment. However, in that study, the immuneresponse-mediated mechanisms underlying the observed serologic and virologicaleffects were not documented. An increase in the number of CD8+ T cells wasreported, but it was not determined whether these T cells are specific for theHBV peptides loaded onto DCs. Furthermore, it has been reported that the core18-27 peptide used to load DCs frequently induces T cell responses in HLA-A0201Caucasian patients, but is rarely able to induce CD8+ T cells in HLA-A0206 orHLA-A0203 Chinese patients [65]. Moreover, the sequences of the two peptidesused in the former study vary according to HBV genotype, raising questions aboutthe specificity of this DC-mediated therapy.
作为疫苗的替换方案，在HBV抗原之后装载DCs可能也可以用于激发T细胞（表二）。使用自身获取的（autologous）DCs和调节癌症-相关的抗原振荡混合（pulsed）一起作为癌症的免疫治疗方法已经被大量的记录【59】。少量的研究报告记录使用DCs细胞治疗病毒相关的感染【60】【61】。
在对五位慢性乙肝感染患者的一个前期试探性的实验，用于评价HbsAg-导致的自身获取的DCs细胞的安全性【62】。单核白血球（Monocyte）衍生的DCs首先用传统的有粒细胞（granulocyte）巨噬细胞克隆刺激因子和IL-4方法从病人的外周血中分离出来。这些细胞然后和HbsAg一起混合振荡，然后再注射到病人体内。这种方法以前显示可以对疫苗不响应的患者导致抗-Hbs的抗体产生【63】。在慢性乙肝患者中，实验是安全的而且没有任何加剧肝脏损伤（exacerbation）。再另一项最近的实验中，自身获取的DCs和从HbcAg（核18-27抗原受体）和病毒中间包膜蛋白的N端得到的HLA-A2-限制性多肽一起振荡混合，然后分9次以一种自身获取的方式，再注射回到慢性乙肝患者中【64】。有趣的是，一个显著的反应可以由以下来证实，一半的HbeAg阴性/抗Hbe阳性患者显示HBVDNA水平下降低于10^3cp/ml, 转氨酶正常化，然而仅仅有13% HbeAg阳性患者显示完全的响应。这项治疗在HbeAg阴性患者中病毒数量<10^5 cp/ml 非常成功，他们中的某些人清除了HbsAg。在前几周的治疗中，有些患者出现了短暂的ALT水平升高，可能反应了治疗方法在受感染的肝细上面的作用，但是alt在治疗的后期回到正常水平。然而，在那项研究中，血清学和病毒学效果下的免疫调节机制并没有记载。有报道说CD8+的T细胞有增加，但是不能确定的是这些T细胞是不是特异性的针对DCs上的HBV多肽。此外，据报道在HLA-A0201白种人（Caucasian）病人身上用于装载DCs的核心18-27多肽频繁的导致T细胞响应，但是在HLA-A0206或者HLA-A0203中国病人身上，却很少能产生CD8+T细胞【65】。另外，由于HBV的基因型的不同，前两项试验的多肽的序列也不同，提出了DC调节的治疗的特异性的问题。
As an alternative to vaccination, DCs loaded ex vivo with HBVantigens could also be used to stimulate T cells (Table 2). The use ofautologous DCs pulsed with tumor-related antigens as immunotherapy for cancerhas been largely documented [59]. Fewstudies have reported the use of DCs for diseases related to viral infections[60], [61]. A pilot study was carried out in five patients with chronic hepatitisB, to evaluate the safety of HBsAg-pulsed autologous DCs [62]. Monocyte-derivedDCs were isolated from peripheral blood from a given patient by a classicalmethod using granulocyte macrophage colony-stimulating factor and IL-4. Theywere then pulsed with purified HBsAg and re-injected into the patient. This approachhad previously been shown to induce anti-HBs antibodies when administered to vaccine-non-responders[63]. Administration was safe and no exacerbation of liver damage was observedin patients with chronic hepatitis B. In another recent trial, autologous DCs pulsedwith HLA-A2-restricted peptides derived from HBcAg (core 18-27 epitope) andfrom the N-terminal part of the middle envelope protein were re-injected on 9occasions into 380 chronic hepatitis B patients in an autologous manner [64].Interestingly, a significant response, as demonstrated by a decrease in HBV DNAlevels to below 103cp/ml and transaminase normalization, was observed in halfthe HBeAg-negative/anti-HBe-positive patients, whereas only 13% of theHBeAg-positive patients displayed complete responses. This treatment was mostsuccessful in HBeAg-negative patients and patients with a viral load <105cp/ml, some of whom had cleared HBsAg. A transient increase in ALT levels was observedin some patients during the first few weeks of treatment, possibly reflectingan effect of the treatment on infected hepatocytes, but ALT levels returned tonormal by the end of treatment. However, in that study, the immuneresponse-mediated mechanisms underlying the observed serologic and virologicaleffects were not documented. An increase in the number of CD8+ T cells wasreported, but it was not determined whether these T cells are specific for theHBV peptides loaded onto DCs. Furthermore, it has been reported that the core18-27 peptide used to load DCs frequently induces T cell responses in HLA-A0201Caucasian patients, but is rarely able to induce CD8+ T cells in HLA-A0206 orHLA-A0203 Chinese patients [65]. Moreover, the sequences of the two peptidesused in the former study vary according to HBV genotype, raising questions aboutthe specificity of this DC-mediated therapy.
作为疫苗的替换方案，在HBV抗原之后装载DCs可能也可以用于激发T细胞（表二）。使用自身获取的（autologous）DCs和调节癌症-相关的抗原振荡混合（pulsed）一起作为癌症的免疫治疗方法已经被大量的记录【59】。少量的研究报告记录使用DCs细胞治疗病毒相关的感染【60】【61】。
在对五位慢性乙肝感染患者的一个前期试探性的实验，用于评价HbsAg-导致的自身获取的DCs细胞的安全性【62】。单核白血球（Monocyte）衍生的DCs首先用传统的有粒细胞（granulocyte）巨噬细胞克隆刺激因子和IL-4方法从病人的外周血中分离出来。这些细胞然后和HbsAg一起混合振荡，然后再注射到病人体内。这种方法以前显示可以对疫苗不响应的患者导致抗-Hbs的抗体产生【63】。在慢性乙肝患者中，实验是安全的而且没有任何加剧肝脏损伤（exacerbation）。再另一项最近的实验中，自身获取的DCs和从HbcAg（核18-27抗原受体）和病毒中间包膜蛋白的N端得到的HLA-A2-限制性多肽一起振荡混合，然后分9次以一种自身获取的方式，再注射回到慢性乙肝患者中【64】。有趣的是，一个显著的反应可以由以下来证实，一半的HbeAg阴性/抗Hbe阳性患者显示HBVDNA水平下降低于10^3cp/ml, 转氨酶正常化，然而仅仅有13% HbeAg阳性患者显示完全的响应。这项治疗在HbeAg阴性患者中病毒数量<10^5 cp/ml 非常成功，他们中的某些人清除了HbsAg。在前几周的治疗中，有些患者出现了短暂的ALT水平升高，可能反应了治疗方法在受感染的肝细上面的作用，但是alt在治疗的后期回到正常水平。然而，在那项研究中，血清学和病毒学效果下的免疫调节机制并没有记载。有报道说CD8+的T细胞有增加，但是不能确定的是这些T细胞是不是特异性的针对DCs上的HBV多肽。此外，据报道在HLA-A0201白种人（Caucasian）病人身上用于装载DCs的核心18-27多肽频繁的导致T细胞响应，但是在HLA-A0206或者HLA-A0203中国病人身上，却很少能产生CD8+T细胞【65】。另外，由于HBV的基因型的不同，前两项试验的多肽的序列也不同，提出了DC调节的治疗的特异性的问题。
In addition to treatments designed to stimulate HBV-specific T cellsin vivo by vaccination strategies or ex vivo with cytokines or DCs, a novelapproach is based on the genetic modification of T cells by T-cell receptor(TCR) gene transfer [69]. This involves (1) isolation of the HBV-specific TCRfrom T cells obtained from individuals with self-resolved hepatitis, (2) theinsertion of this TCR into a DNA or viral vector and (3) the transfer of this TCRspecificity to T cells from patients with chronic HBV infection. Retroviralvectors have been used to introduce HLA-A2-restricted HBV-specific TCR into Tcells from chronic patients. The TCR-transduced T cells produced IFN-γ, TNF-α andIL-2 following target cell recognition and lysed hepatocyte-like cell linesexpressing cognate HBV antigens. In vivo functionality was also assessed by theadoptive transfer of TCR-redirected T cells in immunodeficient mice, leading tothe rejection of grafted tumor cells expressing HBV antigens. This strategyshould make it possible to reconstitute functional HBV-specific T cells inpatients with chronic infection [28, 70]. Alternatively, artificial chimericTCRs composed of a single-chain antibody fragment recognizing HBV envelopeproteins, and cytoplasmic regions of the costimulatory CD28 molecule followedby the CD3-ζ signaling domain have
用于设计激发HBV特异性的T细胞可以在活体中通过疫苗策略，或者在活体之外中利用细胞因子或DCs，此外还有一个基于基因转移T细胞的受体（TCR）基因来达到修改T细胞的新颖方法【69】。这包括（1）从自愈的患者身上的T细胞中分离HBV特异性的TCR，（2）将这段TCR放入到一个DNA或者病毒载体里面（3）把这些TCR转译到慢性HBV携带者身上的T细胞内。逆转录病毒载体已经用于把HLA-A2限制性的HBV特异性的TCR引入到慢性患者的T细胞内。经TCR变换过的T细胞，在识别靶位细胞后，可以生产IFN-γ，TNF-α和IL-2并且可以溶解掉表达HBV同源的抗原的类肝细胞的细胞群。在活体中，其功能通过接受性的转移TCR-从新导向过的T细胞到有免疫缺陷的小鼠身上得到评价，可以导致排除小鼠内移植的表达HBV抗原的癌细胞。这些策略让在慢性患者身上重组功能性的HBV特异性的T细胞成为可能【28,70】。此外，临床由识别HBV膜蛋白的单链抗体片段和细胞液区域的共刺激CD28分子，另外还有CD3 信号区域组成的人工嵌合（chimeric）TCRs被构造。这些嵌合的受体，当被逆转录病毒传递并在细胞表面表达的时候，使人的主要T细胞识别HbsAg阳性的肝细胞，释放IFN-γ和IL-2，更为重要的是，溶解掉含有HBV复制的细胞【71】。然而，是否这些从新注入的TCR-从新导向的T细胞在体内可以扩增和可以接触到靶位器官还不清楚，是否新处理过的T细胞是否会处于一个可容忍（tolerizing）环境。
If anti-HBV immunotherapy is to be efficient, the activated T-cellresponse must specifically reach the liver. Deng et al. [72] therefore designeda recombinant HBV (rHBV) containing a modified viral core gene for the specificdelivery of foreign (i.e. non-HBV) epitopes to the liver. This recombinantvirus was engineered to self-maintain only in hepatocytes already infected withHBV through capsid complementation. The activated non-HBV specific CD8+ T-cellresponse is expected to be functional and to compensate for the deficiencies ofHBV-specific anti-viral immunity. It should target infected liver cells and wouldnot be subject to functional exhaustion during chronic hepatitis B.Proof-of-concept for this approach was demonstrated through a protocol forrHBV-based active immunization in HLA-A2/DR1-transgenic mice (see Fig 3).
如果抗HBV免疫疗法有效，激活的T细胞的响应必须特异性的抵达肝部。Deng et al. [72]因此设计了一个从组的HBV（rHBV）包含一个修改过的病毒核基因用于特异性的传递外来（比如说，非HBV）抗原表位到肝脏。这种组合过的病毒被处理成仅能够在已经被HBV感染的肝细胞内可以自我维持，因为它可以共享HBV的外壳。激活的非HBV特异性的CD8+T细胞响应被认为是功能性的而且可以作为HBV特异性的抗病毒免疫丧失的补充。它应该以受感染的肝细胞为靶位，应该不会导致慢性乙肝的功能性耗竭。为了测试这个概念，一个基于rHBV激活免疫的实验方案选择在HLA-A2/DR1-转基因小鼠上完成了。
（见图 Fig3）
[attach]220460[/attach]
图三:慢性乙肝的基因治疗一个免疫治疗的方案成立了，结合了疫苗和肝脏基因治疗。
设计了一个重组的HBV病毒（rHBV），即作为基因传递的载体也是一个抗原携带物，为在肝内表达一种外来抗原的多重表位。在这项策略中，在使用rHBV基因治疗之前，患者体内首先产生功能性的多表位特异性的T细胞响应。rHBV被构造，通过插入一段外来抗原序列来破坏rHBV病毒的核基因。这个核缺失的病毒没有复制的能力，除非宿主的肝细胞可以反向提供野生型的病毒衣壳蛋白。rHBV特异性的感染人的肝部细胞，和野生型的病毒（wtHBV）一样。然而，rHBV不能再健康的患者中复制，仅能够活在肝细胞长期被野生型wtHBV感染的慢性感染患者身上。在有HBV 肝脏特异性的启动因素出现的时候，rHBV蛋白（聚合酶，L,M,S 包膜蛋白，Hbx）同时还有外来抗原性的多表位被产生，合成并以多肽的形式存在肝细胞内。这些外来的抗原表位可以招来很强的功能性的针对肝细胞的T细胞响应，这些响应可以弥补慢性感染患者身上的有缺陷的HBV特异性的T细胞响应。
A strong polyepitope-specific T-cell response was first primed inthe periphery by DNA immunization. In absence of a mouse model susceptible toHBV infection, hydrodynamic injection was used to mimic rHBV replication andgene expression in the mouse liver. This method allows direct in vivotransfection of hepatocytes and bypass the strict host-range of the virus forhuman hepatocytes [73]. The expression of
foreign antigenic epitopes in hepatocytesrecruited a vigorous T-cell response in situ. Most liver-infiltratingpolyepitope-specific CD8+ T cells proved to be functional effectors. FollowingDNA priming and hydrodynamic injection, the rHBV-based expression of HBsAg inmouse liver was completely inhibited without major liver injury. Studies inHLAA2/DR1/HBsAg-transgenic mice as a surrogate model for HBV chronic infectionfurther validated this approach. In these mice the polyepitope-specific functionalT cell response not only controlled the rHBV expression but also controlled HBVtransgene expression in the liver. Thus, rHBV and foreign antigen-based activeimmunotherapy constitute a promising strategy for the treatment of persistentHBV infection. This strategy could potentially be generalized and extended toother chronic viral diseases.
在外围，由于DNA的免疫，一个很强的多表位特异性的T细胞响应首先发生。在缺少一个易于（susceptible）被HBV感染的老鼠模型，水动力注射被用于模拟在老鼠肝部rHBV的复制和基因表达。这个方法允许直接的活体转染肝细胞并且让这种有严格宿主范围的病毒感染人的肝细胞【73】。由于在肝部表达的外来抗原的表位导致了一个非常强的原位（in situ）T细胞响应。大部分肝部渗透（infiltrating）的多抗原表位特异的CD8+T细胞被证明是功能性的效应器。在DNA 结合和水动力注射后，基于rHBV的老鼠肝部的HBsAg表达被彻底的抑制了，而且肝部没有主要的伤害。在HLAA2/DR1/HbsAg转基因小鼠的研究可以作为代表性的HBV慢性感染模型用于进一步的核实这种方案。在这些老鼠中，多抗原表位特异性的功能T细胞的反应不仅可以控制rHBV的表达，同时也可以控制HBV在转基因小鼠肝部的表达。因此，rHBV和外来抗原激活的免疫治疗组成了一个有希望的策略用于治疗持续的HBV感染。这种方法有潜力扩展和一般化到其他的慢性病毒导致的疾病。
For chronic infections (with HBV, HCV, HIVetc.), therapeutic interventions aim to counteract the negative effects of theimmunosuppressive environment and high antigen load on T cells. The combinationof therapeutic vaccination with other types of immunotherapy and classicalantiviral treatments may prove the most effective strategy for additive or synergisticefficacy. However, the natural course of the disease, the duration of infectionand the immune tolerance status of the targeted patients must be taken intoaccount. Most of the early therapeutic vaccine trials were performed inpatients with persistent infection over several decades, patients with highviral loads or patients who did not respond to classical antiviral treatments.This may accounted for efficacy being lower than initially expected. Indeed,most of the observed beneficial effects concerned patients with HBV viral loadsbelow 106 cp/ml, patients with high ALT levels and HBeAg-negative patients. Thedefinition of subgroups of patients on the basis of biomarkers predictive ofthe efficacy of therapeutic vaccination or other immunotherapy-based strategieswould make it possible to advance toward more sophisticated combinations. Thereconstitution of virus-specific T-cell responses is an important safety issuethat must be carefully evaluated before each new therapeutic intervention. Manyobstacles remain to be overcome before the ideal therapeutic vaccine can bedeveloped on a large scale. However, a growing understanding of the mechanisms underlyingthe immune defects occurring during chronic HBV infection and the experimentaldata collected in previous clinical studies in patients suggest that this “HolyGrail” of modern medicine may be attainable.
对于慢性感染者（包括HBV，HCB，HIV等），治疗性的插入目的是为了抵消免疫抑制的环境带来的消极作用和T细胞上面的高的抗原载量。将治疗性疫苗和其他的免疫疗法还有传统的抗病毒疗法结合起来可能是最有效的策略因为有添加效果或者是协同效果。然而，目标患者的疾病的自然发展，感染持续的时间，免疫耐受的状态都必须考虑进来。大部分的早期的治疗性的疫苗都针对数十年感染的慢性患者，患者有高的病毒载量或者是患者对传统的抗病毒治疗没有响应。这个可以解释为什么治疗性疫苗的效果比预期的要低。事实上，所有观察到的有利的效果都是发生在那些病毒的载量在10^6cp/ml以下，高的转氨酶和HbeAg阴性患者。基于生物标记物来定义不同次小组的患者可以预测治疗型疫苗或者是其他基于免疫治疗的策略的有效性有可能可以升级到更多复杂的组合。每一次治疗的介入（intervention）都必须仔细的评价从组病毒特异性的T细胞响应的安全问题。在理想的治疗性疫苗大规模开发出来之前，还有许多障碍仍然需要克服。然而，越来越多的慢性乙肝感染导致的免疫缺陷的背后机制被了解，并且先前从患者临床收集到的数据提示这块现代医学的圣地有可能可以得到。
Key messages/核心信息:
当前的针对免疫介入疗法和治疗型疫苗的有效性的测量方法缺少统一标准。定义病毒学上的和免疫学上的终点（end-point,应该指治疗指标的终点）可以用于改善分析比较所有临床试验得到的结果。Current measurement ofclinical efficacy following immune-intervention or
therapeuticvaccination lack standardization. Definition of virological and immunologicalend-points that could be used across all clinical studies would improvecomparative analysis between studies.
并不是所有的慢性乙肝患者适合治疗介入。仔细的选择患者对基于免疫的治疗非常有利（成年感染，低病毒载量<10^6 cp/ml, 高的转氨酶）可以增加成功率并且让我们可以更好的理解持续清除HBV的一个潜在机制。Not all patients withchronic hepatitis are good candidates for therapeuticintervention. Carefulselection of patients in which the immune-based therapies would be mostbeneficial (patients infected in adulthood, with low viral load <106 cp/ml, high ALT) would increase the rate of success andallow a better understanding of the mechanisms underlying sustained clearanceof HBV.
定义一个更好的免疫参数可以和基于免疫的或者是疫苗治疗的有效率相关已经被保证，这将毫无疑问的改善现有的正在处理的方案。A better definition ofimmune parameters that correlate with the efficacy of immune-based or vaccinetherapy is yet warranted and will undoubtedly improve the current approaches.
lifevendor 发表于 2011-5-30 12:34
Therapeutic vaccines and immune-basedtherapies for thetreatment of chronichepatitis B: perspectives ...
anny8527 发表于 2011-5-30 16:53
都是骗人的 不要理会
lifevendor 发表于 2011-5-30 17:46
回复 anny8527 的帖子
要积极些，每天接近一点点，慢慢的就解决了！尽管还有很多未知，但是不知道那一天突 ...
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